Compounds for the treatment of neurodegenerative diseases

ABSTRACT

Compounds and their pharmaceutically acceptable salts for treatment of synucleinopathies, such as Parkinson&#39;s disease and tauopathies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/002,164 filed Aug. 29, 2013 which was filed under 35 U.S.C. 371and is a U.S. National Stage Application of PCT/US2012/027222 filed Mar.1, 2012, and claims priority to U.S. Provisional Application No.61/448,935, filed Mar. 3, 2011, each entitled “Compounds for theTreatment of Neurodegenerative Diseases”, the contents of which areincorporated herein by reference.

TECHNICAL FIELD

Provided herein are compounds, pharmaceutical compositions, and methodsfor the treatment of neurodegenerative diseases such as Parkinson'sdisease and various tauopathies.

BACKGROUND OF INVENTION

Parkinson's disease (PD) is a neurodegenerative human disordercharacterized clinically by both motor (movement) and non-motorbehavioral dysfunction, and histopathologically by the formation,deposition, accumulation and/or persistence of abnormal fibrillarprotein deposits and/or aggregates. This accumulation of cytoplasmicLewy bodies consisting of fibrils/aggregates of α-synuclein/NAC (non-APcomponent) is believed important in the pathogenesis of PD. Lewy bodiesoccur mostly in the substantia nigra and locus ceruleus sections of thebrain stem and the olfactory bulb, but also, to a lesser extent, inother subcortical and cortical regions of the brain. Because of thisspecific localization in the brain, Lewy bodies interfere with thehealth and integrity of dopaminergic neuronal projections from thesubstantia nigra to the striatum, thus adversely affecting the abilityto initiate, carry out and control voluntary movements. Lewy bodiespresent in these brain regions may also impact the production ofacetylcholine and/or the balance between dopamine and acetylcholine inthe brain, thus causing disruption in perception, thinking and behavioras well as other non-motor symptoms including loss of smell and sleepdisorders.

Dementia with Lewy Bodies (DLB) is a progressive neurodegenerativedisorder characterized by symptoms which display various degrees ofmanifestation. Such symptoms include progressive dementia, Parkinsonianmovement difficulties, hallucinations, and increased sensitivity toneuroleptic drugs. As with Alzheimer's disease (AD), advanced age isconsidered to be a risk factor for DLB, with average onset typicallybetween the ages of 50-85. Twenty percent of all dementia cases arecaused by DLB and over 50% of PD patients develop Parkinson's DiseaseDementia (PDD), a type of DLB. DLB may occur alone or in conjunctionwith other brain abnormalities, including those involved in AD and PD,as mentioned above. Currently, conclusive diagnosis of DLB is madeduring postmortem autopsy.

New agents or compounds able to bind and/or inhibit α-synuclein and/orNAC formation, deposition, accumulation and/or persistence, or disruptpre-formed α-synuclein/NAC fibrils and/or aggregates (or portionsthereof) are regarded as potential therapeutics for the treatment ofParkinson's and related synucleinopathies. Compounds which protectneurons from degeneration and damage associated with Parkinson's andrelated synucleinopathies could also prove useful as therapeutics.

Parkinson's Disease and Synucleinopathies

Parkinson's disease is a neurodegenerative disorder that ispathologically characterized by the presence of intracytoplasmic Lewybodies (Lewy in Handbuch der Neurologie, M. Lewandowski, ed., Springer,Berlin, pp. 920-933, 1912; Pollanen et al., J. Neuropath. Exp. Neurol.52:183-191, 1993), the major components of which are filamentsconsisting of α-synuclein (Spillantini et al., Proc. Natl. Acad. Sci.USA 95:6469-6473, 1998; Arai et al., Neurosci. Lett. 259:83-86, 1999), a140-amino acid protein (Ueda et al., Proc. Natl. Acad. Sci. USA90:11282-11286, 1993). Three dominant mutations in α-synuclein causingincreased tendency to aggregate and resulting in familial early onsetParkinson's disease have been described suggesting that Lewy bodiescontribute mechanistically to the degeneration of neurons in Parkinson'sdisease and related disorders (Polymeropoulos et al., Science276:2045-2047, 1997; Kruger et al., Nature Genet. 18:106-108, 1998;Zarranz et al., Ann. Neurol. 55:164-173, 2004). Recently, in vitrostudies have demonstrated that recombinant α-synuclein can indeed formLewy body-like fibrils (Conway et al., Nature Med. 4:1318-1320, 1998;Hashimoto et al., Brain Res. 799:301-306, 1998; Nahri et al., J. Biol.Chem. 274:9843-9846, 1999; Choi et al., FEBS Lett. 576:363-368, 2004).Most importantly, both the A53T and the E46K Parkinson's disease-linkedα-synuclein mutations accelerate this fibril-forming aggregationprocess, demonstrating that such in vitro studies may have relevance forParkinson's disease pathogenesis. Alpha-synuclein aggregation and fibrilformation fulfills the criteria of a nucleation-dependent polymerizationprocess (Wood et al., J. Biol. Chem. 274:19509-19512, 1999).Alpha-synuclein recombinant protein, and non-Aβ component (known asNAC), which is a 35-amino acid peptide fragment of α-synuclein, bothhave the ability to form fibrils and/or aggregates when incubated at 37°C., and are positive with stains such as Congo red (demonstrating ared/green birefringence when viewed under polarized light) andThioflavin S (demonstrating positive fluorescence) (Hashimoto et al.,Brain Res. 799:301-306, 1998; Ueda et al., Proc. Natl. Acad. Sci. USA90:11282-11286, 1993).

Synucleins are a family of small, presynaptic neuronal proteins composedof α-, β-, and γ-synucleins, of which only α-synuclein aggregates havebeen associated with several neurological diseases (Ian et al., ClinicalNeurosc. Res. 1:445-455, 2001; Trojanowski and Lee, Neurotoxicology23:457-460, 2002). The role of synucleins (and in particular,α-synuclein) in the etiology of a number of neurodegenerative diseaseshas developed from several observations. Pathologically, synuclein wasidentified as a major component of Lewy bodies, the hallmark inclusionsof Parkinson's disease, and a fragment thereof was isolated from amyloidplaques of a different neurological disease, Alzheimer's disease.Biochemically, recombinant α-synuclein was shown to form fibrils and/oraggregates that recapitulated the ultrastructural features ofα-synuclein isolated from patients with dementia with Lewy bodies,Parkinson's disease and multiple system atrophy. Additionally, theidentification of mutations within the α-synuclein gene, albeit in rarecases of familial Parkinson's disease, demonstrated an unequivocal linkbetween synuclein pathology and neurodegenerative diseases. The commoninvolvement of α-synuclein in a spectrum of diseases such as Parkinson'sdisease, dementia with Lewy bodies, multiple system atrophy and the Lewybody variant of Alzheimer's disease has led to the classification ofthese diseases under the umbrella term of “synucleinopathies”.

NAC is a 35 amino acid fragment of α-synuclein that has the ability toform fibrils and/or aggregates either in vitro or as observed in thebrains of patients with Parkinson's disease. The NAC fragment ofα-synuclein is a relatively important therapeutic target as this portionof α-synuclein is believed crucial for formation of Lewy bodies asobserved in all patients with Parkinson's disease, synucleinopathies andrelated disorders.

Currently available therapeutics such as carbidopa/levodopa (Sinemet,Stalevo, Parcopa), dopamine agonists (Apokyn, Parlodel, Neupro, Mirapex,Requip), anticholinergics (Cogentin, Artane), MAO-B inhibitors(Eldepryl, Carbex, Zelapar, Azilect), COMT inhibitors (Comtan, Tasmar),and other medications like Symmetrel and Exelon aim to slow the loss ofdopamine or improve just the symptoms of the patient.

Discovery and identification of new compounds or agents as potentialtherapeutics to arrest fibril and/or aggregate formation, deposition,accumulation and/or persistence of α-synuclein in Parkinson's disease orprovide neuroprotection are desperately sought.

Parkinson's disease α-synuclein fibrils and/or aggregates consist of apredominantly β-pleated sheet structure. Compounds of this inventionhave been shown to be effective in the inhibition of α-synuclein/NACfibril formation and/or aggregates as well as in the disruption ofpre-formed fibrils and/or aggregates, as shown from Examples providedherein. These compounds could serve as therapeutics for Parkinson'sdisease and other synucleinopathies.

Tau is a microtubule associated protein found primarily in neuronalaxons. Tau hyperphosphorylation is a common characteristic of a numberof dementing disorders collectively known as tauopathies, some of whichhave distinct tau pathology combined with other brain pathologies.Tauopathies include Alzheimer's disease (AD), Pick's disease (PiD),progressive supranuclear palsy (PSP), corticobasal degeneration (CBD)and familial frontotemporal dementia/Parkinsonism linked to chromosome17 (FTDP-17), amyotrophic lateral sclerosis/Parkinsonism-dementiacomplex, argyrophilic grain dementia, dementia pugilistic, diffuseneurofibrillary tangles with calcification, progressive subcorticalgliosis and tangle only dementia. (Spillantini, M G and Goedert M, 1998Trends Neurosci. October 21(10):428-33). In AD, tau pathology istypically limited to the neurons while other tauopathies canpathologically exhibit both neuronal and glial tau deposition (Higuchi,M, et al., 2002. Neuropsychopharmacology: The Fifth Generation ofProgress, Chapter 94: Tau protein and tauopathy).

It has recently been postulated that tau protein may link Parkinson'sand Alzheimer's disease (Shulman, J. M. and DeJager, P. L. 2009 NatureGenetics 41(12):1261-1262). This study examined whether any genome wideassociation occurs between the two diseases and found that three genesand two new loci were linked to increased susceptibility.

Physiological phosphorylation of tau regulates the dynamics of theassociation of tau with tubulin, and thereby microtubule stability(Mazanetz. M. P. and Fischer, P. M. 2007. Nature Reviews 6:464-479). Thestabilization of the microtubules in axons ensures that maintain theirfunction for axonal transport, growth and branching (Bulic, B et al.,2009 Angew. Chem. Int. Ed. 48:2-15). Hyperphosphorylation and misfoldingof the tau protein is thought to be the causative factor in abnormalintracellular aggregation leading ultimately to neuronal dysfunction.Protein aggregates have been found to be toxic to neurons.

Abnormal intraneuronal tau aggregation has three basic pathologicalmanifestations; neurofibrillary tangles (NFT's), neuropil threads (NT's)and the argyrophilic dystrophic neurite plaques (Braak, H and Braak, E,Neurobio. of Aging. 1997 18(4):351-357). Structurally, the NFT's areprincipally comprised of paired helical filaments (PHF) comprised of twofilamentous tau proteins twisted around one another with a crossoverrepeat of 80 nm and a width of 8-20 nm (Li, D., et al., 2008.Computational Biology 4(12) and Kidd, M 1963 Nature, 197:192). There aresix stages (Braak stages I-VI) of tau deposition in the brain, whichprogress temporally at defined anatomical locations with the initialstages characterized primarily by the deposition of NFT's and NT's andthe secondary stages further accompanied by NP (Braak, 1997). InAlzheimers Disease and other neuropathies, Braak's stages correlate wellwith clinical disease progression as demonstrated by increasingcognitive dysfunction. Severe cortical destruction which occurs aroundstages III-IV coincides with the first manifestations of the clinicalonset of AD. Although no tau mutations have been identified in AD thereis a strong correlation between NFT density and cognitive decline in AD(Brunden, K. R., Trojanowski, J. Q., and Lee, V. M. 2009 Nature Reviews8:783-93).

New biomarkers and models of their temporal characteristics are becomingeven more useful for the diagnosis and characterization of AD (Jack etal., 2010. Lancet 9:119-28). Specifically, tau deposition is associatedwith neurodegeneration in AD and an increase in CSF tau is an importantindicator of tau pathologic changes and correlates well with clinicaldisease severity. A decrease in FDG-PET correlates well with increasedCSF tau and both are valid indicators of synaptic dysfunction (Jack etal, ibid). This model of biomarker ordering, especially in mildlycognitive impaired individuals, has important implications for clinicaltrials. Potential therapeutics could be more accurately assessed forefficacy is they are able to change the trajectory of cognitivedeterioration and individuals might be more selectively chosen fortrials (Jack et al, ibid).

It is presently not known if tau is a causative factor in disease but itis likely that either a loss or gain for function results in pathology.In FTLD17, a missense mutation affects the alternative splicing of tauresulting in the disruption of the ratio of the 4R to 3R tau isoform.More of the 4R isoform with an extra repeat of the microtubule bindingregion may lead to overstabilization of the microtubules resulting indisease. Other post-translational events such as alterations in kinaseactivity and glycosylation could also cause hyperphosphorylation andresult in disease or alternatively proteolytic cleavage could producetruncated tau products more inclined to aggregate (Brunden, ibid).

Recently tau toxicity has been re-emphasized as an important therapeutictarget in neurodegerative tauopathies (Keystone Symposium, March 2009).Routes for developing therapeutics are either directed to inhibitingtau-phosphorylation kinases or seeking compounds effective in themodulation of tau aggregation and/or the dissolution or disruption oftau aggregates which may prove equally useful or more specific for thealleviation of tauopathies (Rafii, M. and Aisen, P. 2009 BMC Medicine7:7). A recent paper surveyed the efficacy of several classes ofcompounds for their ability to prevent tau aggregation and disaggregatepre-formed tau fibrils (Bulic et al.). Although there are generalconcerns regarding the toxicity of disassembled fibrils, Bulic et al.,were able to show that reversing tau aggregation resulted in increasedcell viability.

SUMMARY OF INVENTION

In a first aspect, provided herein are compounds such as, but notlimited to:

In a second aspect, this invention is a method of treating asynucleinopathy in a mammal, especially a human, by administration of atherapeutically effective amount of a compound of the first aspect ofthis invention, for example as a pharmaceutical composition. Methodsusing such compounds and compositions for disrupting, disaggregating andcausing removal, reduction or clearance of α-synuclein fibrils and/oraggregates are provided thereby providing new treatments forsynucleinopathies. The treatment of disease may also include theinhibiting the formation of α-synuclein fibrils and/or aggregates orproviding neuroprotection for neurons at risk.

Also provided are any pharmaceutically-acceptable derivatives of thecompounds of the first aspect of this invention, including salts,esters, enol ethers or esters, acetals, ketals, orthoesters,hemiacetals, hemiketals, solvates, hydrates or prodrugs of thecompounds. Pharmaceutically-acceptable salts, include, but are notlimited to, amine salts, alkali metal salts, such as but not limited tolithium, potassium and sodium, alkali earth metal salts, such as but notlimited to barium, calcium and magnesium, transition metal salts, suchas but not limited to zinc and other metal salts, such as but notlimited to sodium hydrogen phosphate and disodium phosphate, and alsoincluding, but not limited to, salts of mineral acids, such as but notlimited to hydrochlorides and sulfates, salts of organic acids, such asbut not limited to acetates, lactates, malates, tartrates, citrates,ascorbates, succinates, butyrates, valerates and fumarates.

Pharmaceutical formulations for administration by an appropriate routeand means containing effective concentrations of one or more of thecompounds provided herein or pharmaceutically acceptable derivatives,such as salts, esters, enol ethers or esters, acetals, ketals,orthoesters, hemiacetals, hemiketals, solvates, hydrates or prodrugs, ofthe compounds that deliver amounts effective for the treatment ofsynucleinopathies, are also provided.

The formulations are compositions suitable for administration by anydesired route and include solutions, suspensions, emulsions, tablets,dispersible tablets, pills, capsules, powders, dry powders forinhalation, sustained release formulations, aerosols for nasal andrespiratory delivery, patches for transdermal delivery and any othersuitable route. The compositions should be suitable for oraladministration, parenteral administration by injection, includingsubcutaneously, intramuscularly or intravenously as an injectableaqueous or oily solution or emulsion, transdermal administration andother selected routes.

Also provided are methods for treatment, prevention or amelioration ofone or more symptoms of synucleinopathies, including but not limited todiseases associated with the formation, deposition, accumulation, orpersistence of alpha-synuclein.

Provided are methods for treatment, prevention or amelioration of one ormore symptoms of synuclein diseases or synucleinopathies. In oneembodiment, the methods inhibit or prevent α-synuclein/NAC fibrilformation and/or aggregation, inhibit or prevent α-synuclein/NAC fibrilgrowth, and/or cause disassembly, disruption, and/or disaggregation ofpreformed α-synuclein/NAC fibrils and α-synuclein/NAC-associated proteindeposits and/or aggregates. Synuclein diseases include, but are notlimited to Parkinson's disease, PDD, familial Parkinson's disease, Lewybody disease, the Lewy body variant of Alzheimer's disease, dementiawith Lewy bodies (DLB), multiple system atrophy, and theParkinsonism-dementia complex of Guam.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating that a compound of the invention causes adose-dependent inhibition of α-synuclein aggregation and fibrilformation as assessed by Thioflavin T fluorometry.

FIG. 2 is a graph illustrating that a compound of the invention causesthe dose-dependent inhibition of α-synuclein aggregation and fibrilformation as assessed by Congo Red binding.

FIG. 3 is a graph illustrating the rotenone dose-dependent increase inThioflavin S fluorescence in BE-M17 neuroblastoma cells overexpressingA53T-mutant α-synuclein.

FIG. 4 is a graph illustrating that a compound of the invention causesdose-dependent inhibition of rotenone-induced cell death as assessed bythe XTT cell viability assay.

FIG. 5 is a graph illustrating that a compound of the invention causesthe dose-dependent inhibition of α-synuclein β-sheet formation asassessed by circular dichroism spectroscopy.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications are incorporated byreference in their entirety. In the event that there is a plurality ofdefinitions for a term herein, those in this section prevail unlessstated otherwise.

As used herein, “Synuclein diseases” or “synucleinopathies” are diseasesassociated with the formation, deposition, accumulation, or persistenceof synuclein fibrils, including, but not limited to α-synuclein fibrils.Such diseases include, but are not limited to Parkinson's disease,Familial Parkinson's disease, PDD, Lewy body disease, the Lewy bodyvariant of Alzheimer's disease, dementia with Lewy bodies, multiplesystem atrophy, and the Parkinsonism-dementia complex of Guam.

Aggregation or Fibrillogenesis refers to the formation, deposition,accumulation and/or persistence of synuclein fibrils, filaments,inclusions, deposits, and/or NAC fibrils, filaments, inclusions,deposits, and/or aggregates or the like.

Inhibition of aggregation or fibrillogenesis refers to the inhibition offormation, deposition, accumulation and/or persistence of such fibrilsor fibril-like deposits.

Disruption of fibrils or fibrillogenesis refers to the disruption ofpre-formed α-synuclein fibrils, that usually exist in a pre-dominantβ-pleated sheet secondary structure. Such disruption by compoundsprovided herein may involve marked reduction or disassembly of synucleinfibrils as assessed by various methods such as Thioflavin T fluorometry,Congo red binding, SDS-PAGE/Western blotting, as demonstrated by theExamples presented in this application.

“Mammal” includes both humans and non-human mammals, such as companionanimals (cats, dogs, and the like), laboratory animals (such as mice,rats, guinea pigs, and the like) and farm animals (cattle, horses,sheep, goats, swine, and the like).

“Pharmaceutically acceptable excipient” means an excipient that isconventionally useful in preparing a pharmaceutical composition that isgenerally safe, non-toxic, and desirable, and includes excipients thatare acceptable for veterinary use or for human pharmaceutical use. Suchexcipients may be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

A “therapeutically effective amount” means the amount that, whenadministered to a subject or animal for treating a disease, issufficient to affect the desired degree of treatment, prevention orsymptom amelioration for the disease. A “therapeutically effectiveamount” or a “therapeutically effective dosage” in certain embodimentsinhibits, reduces, disrupts, disassembles synuclein fibril formation,deposition, accumulation and/or persistence, or treats, prevents, orameliorates one or more symptoms of a disease associated with theseconditions, such as a synucleinopathy, in a measurable amount in oneembodiment, by at least 20%, in other embodiment, by at least 40%, inother embodiment by at least 60%, and in still other embodiment by atleast 80%, relative to an untreated subject. Effective amounts of acompound provided herein or composition thereof for treatment of amammalian subject are about 0.1 to about 1000 mg/Kg of body weight ofthe subject/day, such as from about 1 to about 100 mg/Kg/day, in otherembodiment, from about 10 to about 100 mg/Kg/day. A broad range ofdisclosed composition dosages are believed to be both safe andeffective.

The term “sustained release component” is defined herein as a compoundor compounds, including, but not limited to, polymers, polymer matrices,gels, permeable membranes, liposomes, microspheres, or the like, or acombination thereof, that facilitates the sustained release of theactive ingredient.

If the complex is water-soluble, it may be formulated in an appropriatebuffer, for example, phosphate buffered saline, or other physiologicallycompatible solutions. Alternatively, if the resulting complex has poorsolubility in aqueous solvents, then it may be formulated with anon-ionic surfactant such as Tween, or polyethylene glycol. Thus, thecompounds and their physiologically suitable solvents may be formulatedfor administration by inhalation or insufflation (either through themouth or the nose) or oral, buccal, parenteral, or rectaladministration, as examples.

As used herein, pharmaceutically acceptable derivatives of a compoundinclude salts, esters, enol ethers, enol esters, acetals, ketals,orthoesters, hemiacetals, hemiketals, solvates, hydrates or prodrugsthereof. Such derivatives may be readily prepared by those of skill inthis art using known methods for such derivatization. The compoundsproduced may be administered to animals or humans without substantialtoxic effects and either are pharmaceutically active or are prodrugs.Pharmaceutically acceptable salts include, but are not limited to, aminesalts, such as but not limited to N,N′-dibenzylethylenediamine,chloroprocaine, choline, ammonia, diethanolamine and otherhydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine,N-benzylphenethylamine,1-para-chlorobenzyl-2-pyrrolidin-1′-ylmethyl-benzimidazole, diethylamineand other alkylamines, piperazine and tris(hydroxymethyl)aminomethane;alkali metal salts, such as but not limited to lithium, potassium andsodium; alkali earth metal salts, such as but not limited to barium,calcium and magnesium; transition metal salts, such as but not limitedto zinc; and other metal salts, such as but not limited to sodiumhydrogen phosphate and disodium phosphate; and also including, but notlimited to, salts of mineral acids, such as but not limited tohydrochlorides and sulfates; and salts of organic acids, such as but notlimited to acetates, lactates, malates, tartrates, citrates, ascorbates,succinates, butyrates, valerates and fumarates. Pharmaceuticallyacceptable esters include, but are not limited to, alkyl, alkenyl,alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl andheterocyclyl esters of acidic groups, including, but not limited to,carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids,sulfinic acids and boronic acids. Pharmaceutically acceptable enolethers include, but are not limited to, derivatives of formula C═C(OR)where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl,heteroaralkyl, cycloalkyl or heterocyclyl. Pharmaceutically acceptableenol esters include, but are not limited to, derivatives of formulaC═C(OC(O)R) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl,heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl.Pharmaceutically acceptable solvates and hydrates are complexes of acompound with one or more solvent or water molecules, or 1 to about 100,or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.

As used herein, treatment means any manner in which one or more of thesymptoms of a disease or disorder are ameliorated or otherwisebeneficially altered. For example, slowing or arresting diseasedevelopment, providing relief from the symptoms or side-effects of thedisease, and relieving the disease by causing regression of the disease,such as by disruption of pre-formed synuclein fibrils could all beconsidered as treatment.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular compound or pharmaceutical compositionrefers to any lessening, whether permanent or temporary, lasting ortransient that can be attributed to or associated with administration ofthe composition.

As used herein, “NAC” (non-Aβ component) is a 35-amino acid peptidefragment of α-synuclein, which like α-synuclein, has the ability to formfibrils when incubated at 37° C., and is positive with stains such asCongo red (demonstrating a red/green birefringence when viewed underpolarized light) and Thioflavin S (demonstrating positive fluorescence)(Hashimoto et al., Brain Res. 799:301-306, 1998; Ueda et al., Proc.Natl. Acad. Sci. U.S.A. 90:11282-11286, 1993). Inhibition of NAC fibrilformation, deposition, accumulation, aggregation, and/or persistence isbelieved to be effective treatment for a number of diseases involvingα-synuclein, such as Parkinson's disease, Lewy body disease and multiplesystem atrophy.

As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized by one or more steps or processes orotherwise converted to the biologically, pharmaceutically ortherapeutically active form of the compound. To produce a prodrug, thepharmaceutically active compound is modified such that the activecompound will be regenerated by metabolic processes. The prodrug may bedesigned to alter the metabolic stability or the transportcharacteristics of a drug, to mask side effects or toxicity, to improvethe flavor of a drug or to alter other characteristics or properties ofa drug. By virtue of knowledge of pharmacodynamic processes and drugmetabolism in vivo, those of skill in this art, once a pharmaceuticallyactive compound is known, can design prodrugs of the compound (see,e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, OxfordUniversity Press, New York, pages 388-392).

It is to be understood that the compounds provided herein may containchiral centers. Such chiral centers may be of either the (R) or (S)configuration, or may be a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, or be stereoisomeric ordiastereomeric mixtures. It is to be understood that the chiral centersof the compounds provided herein may undergo epimerization in vivo. Assuch, one of skill in the art will recognize that administration of acompound in its (R) form is equivalent, for compounds that undergoepimerization in vivo, to administration of the compound in its (S)form.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem.11:942-944).

B. Compounds

Provided herein are compounds and pharmaceutical compositions containingcompounds, not limited to those shown above in the summary section andin the following examples.

C. Preparation of the Compounds

The compounds provided herein can be prepared by standard syntheticmethods known in the art, and are shown in general schemes providedherein. The examples that follow describe the exemplary embodiments andare not purported to limit the scope of the claimed subject matter. Itis intended that the specification, together with the followingexamples, be considered exemplary only, with the scope and spirit of theclaimed subject matter being indicated by the claims that follow theseexamples. Other embodiments within the scope of claims herein will beapparent to one skilled in the art from consideration of thespecification as described herein.

The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as the AldrichChemical Company (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma(St. Louis, Mo.), or Lancaster Synthesis Inc. (Windham, N. H.) or areprepared by methods well known to a person of ordinary skill in the art,following procedures described in such references as Fieser and Fieser'sReagents for Organic Synthesis, vols. 1-17, John Wiley and Sons, NewYork, N.Y., 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 andsupps., Elsevier Science Publishers, 1989; Organic Reactions, vols.1-40, John Wiley and Sons, New York, N.Y., 1991; March J.: AdvancedOrganic Chemistry, 4th ed., John Wiley and Sons, New York, N.Y.; andLarock: Comprehensive Organic Transformations, VCH Publishers, New York,1989.

In most cases, protective groups for the hydroxy groups are introducedand finally removed. Suitable protective groups are described in Greeneet al., Protective Groups in Organic Synthesis, Second Edition, JohnWiley and Sons, New York, 1991. Other starting materials or earlyintermediates may be prepared by elaboration of the materials listedabove, for example, by methods well known to a person of ordinary skillin the art. The starting materials, intermediates, and compoundsprovided herein may be isolated and purified using conventionaltechniques, including precipitation, filtration, distillation,crystallization, chromatography, and the like. The compounds may becharacterized using conventional methods, including physical constantsand spectroscopic methods.

D. Pharmaceutical Compositions and Administration

The compounds provided herein can be used as such, be administered inthe form of pharmaceutically acceptable salts derived from inorganic ororganic acids, or used in combination with one or more pharmaceuticallyacceptable excipients. The phrase “pharmaceutically acceptable salt”means those salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues without undue toxicity,irritation, allergic response, and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. The salts can be prepared either in situ duringthe final isolation and purification of the compounds provided herein orseparately by reacting the acidic or basic drug substance with asuitable base or acid respectively. Typical salts derived from organicor inorganic acids salts include, but are not limited to hydrochloride,hydrobromide, hydroiodide, acetate, adipate, alginate, citrate,aspartate, benzoate, bisulfate, gluconate, fumarate, hydroiodide,lactate, maleate, oxalate, palmitoate, pectinate, succinate, tartrate,phosphate, glutamate, and bicarbonate. Typical salts derived fromorganic or inorganic bases include, but are not limited to lithium,sodium, potassium, calcium, magnesium, ammonium, monoalkylammonium suchas meglumine, dialkylammonium, trialkylammonium, and tetralkylammonium.The mode of administration of the pharmaceutical compositions can beoral, rectal, intravenous, intramuscular, intracisternal, intravaginal,intraperitoneal, bucal, subcutaneous, intrasternal, nasal, or topical.The compositions can also be delivered at the target site through acatheter, an intracoronary stent (a tubular device composed of a finewire mesh), a biodegradable polymer, or biological carriers including,but are not limited to antibodies, biotin-avidin complexes, and thelike. Dosage forms for topical administration of a compound providedherein include powders, sprays, ointments and inhalants. The activecompound is mixed under sterile conditions with a pharmaceuticallyacceptable carrier and any needed preservatives, buffers or propellants.Opthalmic formulations, eye ointments, powders and solutions are alsoprovided herein.

Actual dosage levels of active ingredients and the mode ofadministration of the pharmaceutical compositions provided herein can bevaried in order to achieve the effective therapeutic response for aparticular patient. The phrase “therapeutically effective amount” of thecompound provided herein means a sufficient amount of the compound totreat disorders, at a reasonable benefit/risk ratio applicable to anymedical treatment. It will be understood, however, that the total dailyusage of the compounds and compositions of the provided will be decidedby the attending physician within the scope of sound medical judgment.The total daily dose of the compounds provided herein may range fromabout 0.0001 to about 1000 mg/kg/day. For purposes of oraladministration, doses can be in the range from about 0.001 to about 50mg/kg/day. If desired, the effective daily dose can be divided intomultiple doses for purposes of administration; consequently, single dosecompositions may contain such amounts or submultiples thereof to make upthe daily dose. The specific therapeutically effective dose level forany particular patient will depend upon a variety of factors includingthe disorder being treated and the severity of the disorder; medicalhistory of the patient, activity of the specific compound employed; thespecific composition employed, age, body weight, general health, sex anddiet of the patient, the time of administration, route ofadministration, the duration of the treatment, rate of excretion of thespecific compound employed, drugs used in combination or coincidentalwith the specific compound employed; and the like.

The compounds provided can be formulated together with one or morenon-toxic pharmaceutically acceptable diluents, carriers, adjuvants, andantibacterial and antifungal agents such as parabens, chlorobutanol,phenol, sorbic acid, and the like. Proper fluidity can be maintained,for example, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants. In some cases, in order to prolong theeffect of the drug, it is desirable to decrease the rate of absorptionof the drug from subcutaneous or intramuscular injection. This can beaccomplished by suspending crystalline or amorphous drug substance in avehicle having poor water solubility such as oils. The rate ofabsorption of the drug then depends upon its rate of dissolution, which,in turn, may depend upon crystal size and crystalline form. Prolongedabsorption of an injectable pharmaceutical form can be achieved by theuse of absorption delaying agents such as aluminum monostearate orgelatin.

The compound provided herein can be administered enterally orparenterally in solid or liquid forms. Compositions suitable forparenteral injection may comprise physiologically acceptable, isotonicsterile aqueous or nonaqueous solutions, dispersions, suspensions, oremulsions, and sterile powders for reconstitution into sterileinjectable solutions or dispersions. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and thelike), vegetable oils (such as olive oil), injectable organic esterssuch as ethyl oleate, and suitable mixtures thereof. These compositionscan also contain adjuvants such as preserving, wetting, emulsifying, anddispensing agents. Suspensions, in addition to the active compounds, maycontain suspending agents such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,or mixtures of these substances.

The compounds provided herein can also be administered by injection orinfusion, either subcutaneously or intravenously, or intramuscularly, orintrasternally, or intranasally, or by infusion techniques in the formof sterile injectable or oleaginous suspension. The compound may be inthe form of a sterile injectable aqueous or oleaginous suspensions.These suspensions may be formulated according to the known art usingsuitable dispersing of wetting agents and suspending agents that havebeen described above. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilsmay be conventionally employed including synthetic mono- ordiglycerides. In addition fatty acids such as oleic acid find use in thepreparation of injectables. Dosage regimens can be adjusted to providethe optimum therapeutic response. For example, several divided dosagesmay be administered daily or the dosage may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

Injectable dosage forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable medium just prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound may be mixed with at least one inert, pharmaceuticallyacceptable excipient or carrier, such as sodium citrate or dicalciumphosphate and/or (a) fillers or extenders such as starches, lactose,sucrose, glucose, mannitol and silicic acid; (b) binders such ascarboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose and acacia; (c) humectants such as glycerol; (d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates and sodium carbonate; (e) solutionretarding agents such as paraffin; (f) absorption accelerators such asquaternary ammonium compounds; (g) wetting agents such as cetyl alcoholand glycerol monostearate; (h) absorbents such as kaolin and bentoniteclay and (i) lubricants such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate and mixturesthereof. In the case of capsules, tablets and pills, the dosage form mayalso comprise buffering agents. Solid compositions of a similar type mayalso be employed as fillers in soft and hard-filled gelatin capsulesusing such excipients as lactose or milk sugar as well as high molecularweight polyethylene glycols and the like.

The solid dosage forms of tablets, dragees, capsules, pills and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well-known in the pharmaceutical formulating art. Theymay optionally contain opacifying agents and may also be of acomposition such that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Tablets contain thecompound in admixture with non-toxic pharmaceutically acceptableexcipients that are suitable for the manufacture of tablets. Theseexcipients may be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, maizestarch or alginic acid; binding agents, for example, maize starch,gelatin or acacia, and lubricating agents, for example, magnesiumstearate or stearic acid or tale. The tablets may be uncoated or theymay be coated by known techniques to delay disintegration and absorptionin the gastrointestinal tract and thereby provide a sustained actionover a longer period. For example, a time delay material such asglycerol monostearate or glycerol distearate may be employed.Formulations for oral use may also be presented as hard gelatin capsuleswherein the compound is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate or kaolin, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, peanut oil, liquid paraffin or olive oil.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art such as, for example, water orother solvents, solubilizing agents and emulsifiers such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan andmixtures thereof. Besides inert diluents, the oral compositions may alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring and perfuming agents.

Aqueous suspensions contain the compound in admixture with excipientssuitable for the manufacture of aqueous suspensions. Such excipients aresuspending agents, for example, sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethyl cellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents may be naturally occurring phosphatides, for examplelecithin, or condensation products of an alkylene oxide with fattyacids, for example polyoxyethylene stearate, or condensation products ofethylene oxide with long chain aliphatic alcohols, for example,heptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids such as hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters from fatty acids and a hexitolanhydrides, for example, polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives, for example,ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents, or one or more sweetening agents, such as sucroseor saccharin.

Oily suspensions may be formulated by suspending the compound in avegetable oil, for example arachis oil, olive oil, sesame oil, orcoconut oil or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents, such as those set forthbelow, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of anantioxidant such as ascorbic acid. Dispersible powders and granulessuitable for preparation of an aqueous suspension by the addition ofwater provide the active ingredient in admixture with a dispersing orwetting agent, a suspending agent and one or more preservatives.Suitable dispersing or wetting agents and suspending agents areexemplified by those already described above. Additional excipients, forexample sweetening, flavoring and agents, may also be present.

The compounds provided herein may also be in the form of oil-in-wateremulsions. The oily phase may be a vegetable oil, for example olive oilor arachis oils, or a mineral oil, for example liquid paraffin ormixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally occurring phosphatides, for example soy bean, lecithin, andoccurring phosphatides, for example soy bean, lecithin, and esters orpartial esters derived from fatty acids and hexitol anhydrides, forexample sorbitan monooleate, and condensation products of the saidpartial esters with ethylene oxide, for example polyoxyethylene sorbitanmonooleate. The emulsion may also contain sweetening and flavoringagents. Syrups and elixirs may be formulated with sweetening agents, forexample, glycerol, sorbitol or sucrose. Such formulations may alsocontain a demulcent, preservative and flavoring and coloring agent.

In one embodiment, the compounds are formulated in dosage unit form forease of administration and uniformity of dosage. Dosage unit form asused herein refers to physically discrete units suited as unitarydosages for the subjects to be treated; each containing atherapeutically effective quantity of the compound and at least onepharmaceutical excipient. A drug product will comprise a dosage unitform within a container that is labeled or accompanied by a labelindicating the intended method of treatment, such as the treatment of adisease associated with α-synuclein/NAC fibril formation such asParkinson's disease. Compositions for rectal or vaginal administrationare preferably suppositories which can be prepared by mixing thecompounds provided herein with suitable non-irritating excipients orcarriers such as cocoa butter, polyethylene glycol or a suppository waxwhich are solid at room temperature but liquid at body temperature andtherefore melt in the rectum or vaginal cavity and release the activecompound.

Compounds provided herein can also be administered in the form ofliposomes. Methods to form liposomes are known in the art (Prescott,Ed., Methods in Cell Biology 1976, Volume XIV, Academic Press, New York,N.Y.) As is known in the art, liposomes are generally derived fromphospholipids or other lipid substances. Liposomes are formed by mono-or multi-lamellar hydrated liquid crystals which are dispersed in anaqueous medium. Any non-toxic, physiologically acceptable andmetabolizable lipid capable of forming liposomes can be used. Thepresent compositions in liposome form can contain, in addition to acompound provided herein, stabilizers, preservatives, excipients and thelike. The preferred lipids are natural and synthetic phospholipids andphosphatidyl cholines (lecithins).

The compounds provided herein can also be administered in the form of a‘prodrug’ wherein the active pharmaceutical ingredients are released invivo upon contact with hydrolytic enzymes such as esterases andphosphatases in the body. The term “pharmaceutically acceptableprodrugs” as used herein represents those prodrugs of the compoundsprovided herein, which are, within the scope of sound medical judgment,suitable for use in contact with the tissues without undue toxicity,irritation, allergic response, and the like, commensurate with areasonable benefit/risk ratio, and effective for their intended use. Athorough discussion is provided in T. Higuchi and V. Stella (Higuchi, T.and Stella, V. Pro-drugs as Novel Delivery Systems, V. 14 of the A.C.S.Symposium Series; Edward B. Roche, Ed., Bioreversible Carriers in DrugDesign 1987, American Pharmaceutical Association and Pergamon Press),which is incorporated herein by reference.

The compounds provided herein, or pharmaceutically acceptablederivatives thereof, may also be formulated to be targeted to aparticular tissue, receptor, or other area of the body of the subject tobe treated. Many such targeting methods are well known to those of skillin the art. All such targeting methods are contemplated herein for usein the instant compositions. For non-limiting examples of targetingmethods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359,6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082,6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252,5,840,674, 5,759,542 and 5,709,874.

In one embodiment, liposomal suspensions, including tissue-targetedliposomes, such as tumor-targeted liposomes, may also be suitable aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art. For example, liposomeformulations may be prepared as described in U.S. Pat. No. 4,522,811.Briefly, liposomes such as multilamellar vesicles (MLVs) may be formedby drying down egg phosphatidyl choline and brain phosphatidyl serine(7:3 molar ratio) on the inside of a flask. A solution of a compoundprovided herein in phosphate buffered saline lacking divalent cations(PBS) is added and the flask shaken until the lipid film is dispersed.The resulting vesicles are washed to remove unencapsulated compound,pelleted by centrifugation, and then resuspended in PBS.

Sustained Release Formulations

Also provided are sustained release formulations to deliver thecompounds to the desired target (i.e. brain) at high circulating levels(between 10⁻⁹ and 10⁻⁴ M). In a certain embodiment for the treatment ofParkinson's disease, the circulating levels of the compounds aremaintained up to 10⁻⁷ M. The levels are either circulating in thepatient systemically, or in one embodiment, present in brain tissue, andin other embodiments, localized to the α-synuclein fibril deposits inbrain.

It is understood that the compound levels are maintained over a certainperiod of time as is desired and can be easily determined by one skilledin the art. In one embodiment, the administration of a sustained releaseformulation is effected so that a constant level of therapeutic compoundis maintained between 10⁻⁸ and 10⁻⁶M between 48 to 96 hours in the sera.

Such sustained and/or timed release formulations may be made bysustained release means of delivery devices that are well known to thoseof ordinary skill in the art, such as those described in U.S. Pat. Nos.3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384;5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476;5,354,556 and 5,733,566, the disclosures of which are each incorporatedherein by reference. These pharmaceutical compositions can be used toprovide slow or sustained release of one or more of the active compoundsusing, for example, hydroxypropylmethyl cellulose, other polymermatrices, gels, permeable membranes, osmotic systems, multilayercoatings, microparticles, liposomes, microspheres, or the like. Suitablesustained release formulations known to those skilled in the art,including those described herein, may be readily selected for use withthe pharmaceutical compositions provided herein. Thus, single unitdosage forms suitable for oral administration, such as, but not limitedto, tablets, capsules, gelcaps, caplets, powders and the like, that areadapted for sustained release are contemplated herein.

In one embodiment, the sustained release formulation contains activecompound such as, but not limited to, microcrystalline cellulose,maltodextrin, ethylcellulose, and magnesium stearate. As describedabove, all known methods for encapsulation which are compatible withproperties of the disclosed compounds are contemplated herein. Thesustained release formulation is encapsulated by coating particles orgranules of the pharmaceutical compositions provided herein with varyingthickness of slowly soluble polymers or by microencapsulation. In oneembodiment, the sustained release formulation is encapsulated with acoating material of varying thickness (e.g. about 1 micron to 200microns) that allow the dissolution of the pharmaceutical compositionabout 48 hours to about 72 hours after administration to a mammal. Inanother embodiment, the coating material is a food-approved additive.

In another embodiment, the sustained release formulation is a matrixdissolution device that is prepared by compressing the drug with aslowly soluble polymer carrier into a tablet. In one embodiment, thecoated particles have a size range between about 0.1 to about 300microns, as disclosed in U.S. Pat. Nos. 4,710,384 and 5,354,556, whichare incorporated herein by reference in their entireties. Each of theparticles is in the form of a micromatrix, with the active ingredientuniformly distributed throughout the polymer.

Sustained release formulations such as those described in U.S. Pat. No.4,710,384, which is incorporated herein by reference in its entirety,having a relatively high percentage of plasticizer in the coating inorder to permit sufficient flexibility to prevent substantial breakageduring compression are disclosed. The specific amount of plasticizervaries depending on the nature of the coating and the particularplasticizer used. The amount may be readily determined empirically bytesting the release characteristics of the tablets formed. If themedicament is released too quickly, then more plasticizer is used.Release characteristics are also a function of the thickness of thecoating. When substantial amounts of plasticizer are used, the sustainedrelease capacity of the coating diminishes. Thus, the thickness of thecoating may be increased slightly to make up for an increase in theamount of plasticizer. Generally, the plasticizer in such an embodimentwill be present in an amount of about 15 to 30% of the sustained releasematerial in the coating, in one embodiment 20 to 25%, and the amount ofcoating will be from 10 to 25% of the weight of the active material, andin another embodiment, 15 to 20% of the weight of active material. Anyconventional pharmaceutically acceptable plasticizer may be incorporatedinto the coating.

The compounds provided herein can be formulated as a sustained and/ortimed release formulation. All sustained release pharmaceutical productshave a common goal of improving drug therapy over that achieved by theirnon-sustained counterparts. Ideally, the use of an optimally designedsustained release preparation in medical treatment is characterized by aminimum of drug substance being employed to cure or control thecondition. Advantages of sustained release formulations may include: 1)extended activity of the composition, 2) reduced dosage frequency, and3) increased patient compliance. In addition, sustained releaseformulations can be used to affect the time of onset of action or othercharacteristics, such as blood levels of the composition, and thus canaffect the occurrence of side effects.

The sustained release formulations provided herein are designed toinitially release an amount of the therapeutic composition that promptlyproduces the desired therapeutic effect, and gradually and continuallyrelease of other amounts of compositions to maintain this level oftherapeutic effect over an extended period of time. In order to maintainthis constant level in the body, the therapeutic composition must bereleased from the dosage form at a rate that will replace thecomposition being metabolized and excreted from the body.

The sustained release of an active ingredient may be stimulated byvarious inducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. In one embodiment, thecompounds are formulated as controlled release powders of discretemicroparticles that can be readily formulated in liquid form. Thesustained release powder comprises particles containing an activeingredient and optionally, an excipient with at least one non-toxicpolymer.

The powder can be dispersed or suspended in a liquid vehicle and willmaintain its sustained release characteristics for a useful period oftime. These dispersions or suspensions have both chemical stability andstability in terms of dissolution rate. The powder may contain anexcipient comprising a polymer, which may be soluble, insoluble,permeable, impermeable, or biodegradable. The polymers may be polymersor copolymers. The polymer may be a natural or synthetic polymer.Natural polymers include polypeptides (e.g., zein), polysaccharides(e.g., cellulose), and alginic acid. Representative synthetic polymersinclude those described, but not limited to, those described in column3, lines 33-45 of U.S. Pat. No. 5,354,556, which is incorporated byreference in its entirety. Particularly suitable polymers include thosedescribed, but not limited to those described in column 3, line46-column 4, line 8 of U.S. Pat. No. 5,354,556 which is incorporated byreference in its entirety.

The sustained release compositions provided herein may be formulated forparenteral administration, e.g., by intramuscular injections or implantsfor subcutaneous tissues and various body cavities and transdermaldevices. In one embodiment, intramuscular injections are formulated asaqueous or oil suspensions. In an aqueous suspension, the sustainedrelease effect is due to, in part, a reduction in solubility of theactive compound upon complexation or a decrease in dissolution rate. Asimilar approach is taken with oil suspensions and solutions, whereinthe release rate of an active compound is determined by partitioning ofthe active compound out of the oil into the surrounding aqueous medium.Only active compounds which are oil soluble and have the desiredpartition characteristics are suitable. Oils that may be used forintramuscular injection include, but are not limited to, sesame, olive,arachis, maize, almond, soybean, cottonseed and castor oil.

A highly developed form of drug delivery that imparts sustained releaseover periods of time ranging from days to years is to implant adrug-bearing polymeric device subcutaneously or in various bodycavities. The polymer material used in an implant, which must bebiocompatible and nontoxic, include but are not limited to hydrogels,silicones, polyethylenes, ethylene-vinyl acetate copolymers, orbiodegradable polymers.

Article of Manufacture

The compounds or pharmaceutically acceptable derivatives may be packagedas articles of manufacture containing packaging material, a compound orpharmaceutically acceptable derivative thereof provided herein, which iseffective for treatment, prevention or amelioration of one or moresymptoms of synuclein diseases, within the packaging material, and alabel that indicates that the compound or composition, orpharmaceutically acceptable derivative thereof, is used for treatment,prevention or amelioration of one or more symptoms of synucleindiseases. The articles of manufacture provided herein contain packagingmaterials. Packaging materials for use in packaging pharmaceuticalproducts are well known to those of skill in the art. See, e.g., U.S.Pat. Nos. 5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceuticalpackaging materials include, but are not limited to, blister packs,bottles, tubes, inhalers, pumps, bags, vials, containers, syringes,bottles, and any packaging material suitable for a selected formulationand intended mode of administration and treatment. A wide array offormulations of the compounds and compositions provided herein arecontemplated as are a variety of treatments for synuclein diseases.

E. Evaluation of the Activity of the Compounds

The biological activity of the compounds provided herein asdisruptors/inhibitors of Parkinson's disease α-synuclein fibrils wasassessed by determining the efficacy of the compounds to cause adisassembly/disruption of pre-formed Parkinson's disease α-synucleinfibrils. In one study, Thioflavin T fluorometry was used to determinethe effects of the compounds, and of anegative control referencecompound). In this assay Thioflavin T binds specifically to fibrillarprotein, and this binding produces a fluorescence enhancement at 485 nmthat is directly proportional to the amount of fibrils present. Thehigher the fluorescence, the greater the amount of fibrils present (Nakiet al, Lab. Invest. 65:104-110, 1991; Levine III, Protein Sci.2:404-410, 1993; Amyloid: Int. J. Exp. Clin. Invest. 2:1-6, 1995).

In the Congo red binding assay the ability of a given test compound toalter α-synuclein fibril binding to Congo red was quantified. In thisassay, α-synuclein fibrils and test compounds were incubated for 2 daysand then vacuum filtered through a 0.2 μm filter. The amount ofα-synuclein fibrils retained in the filter was then quantitatedfollowing staining of the filter with Congo red. After appropriatewashing of the filter, any lowering of the Congo red color on the filterin the presence of the test compound (compared to the Congo red stainingof the protein in the absence of the test compound) was indicative ofthe test compound's ability to diminish/alter the amount of aggregatedand congophilic α-synuclein fibrils.

F. Combination Therapy

In another embodiment, the compounds may be administered in combination,or sequentially, with another therapeutic agent. Such other therapeuticagents include those known for treatment, prevention, or amelioration ofone or more symptoms of synuclein diseases. Such therapeutic agentsinclude, but are not limited to; carbidopa/levodopa (Sinemet, Stalevo,Parcopa), dopamine agonists (Apokyn, Parlodel, Neupro, Mirapex, Requip),anticholinergics (Cogentin, Artane), MAO-B inhibitors (Eldepryl, Carbex,Zelapar, Azilect), COMT inhibitors (Comtan, Tasmar), and othermedications like Symmetrel and Exelon.

G. Methods of Use of the Compounds and Compositions

The compounds and compositions provided herein are useful in methods oftreatment, prevention, or amelioration of one or more symptoms ofsynucleopathies, including but not limited to diseases associated withthe formation, deposition, accumulation, or persistence of synucleinfibrils. Also provided are methods to inhibit or prevent α-synuclein/NACfibril formation and/or aggregation, methods to inhibit or preventα-synuclein/NAC fibril growth, and methods to cause disassembly,disruption, and/or disaggregation of preformed α-synuclein/NAC fibrilsand α-synuclein/NAC-associated protein deposits.

In certain embodiments, the synuclein diseases or synucleinopathiestreated, prevented or whose symptoms are ameliorated by the compoundsand compositions provided herein include, but are not limited todiseases associated with the formation, deposition, accumulation, orpersistence of synuclein fibrils, including α-synuclein fibrils and/oraggregates. In certain embodiments, such diseases include Parkinson'sdisease, PDD, familial Parkinson's disease, Lewy body disease, the Lewybody variant of Alzheimer's disease, dementia with Lewy bodies, multiplesystem atrophy, and the Parkinsonism-dementia complex of Guam.

The following non-limiting Examples are given by way of illustrationonly and are not considered a limitation of the subject matter, manyapparent variations of which are possible without departing from thespirit or scope thereof.

EXAMPLES Example 1 Synthesis of SA-52

3,4-dimethoxybenzoic acid (1) (1.00 g, 5.5 mmol) was slurried in 5 mL ofDCM. Oxalyl chloride (0.9 mL, 10.5 mmol) was then added, and five dropsof DMF were added to initiate the reaction. The mixture was stirredovernight during which time it became a yellow solution. The solutionwas concentrated and dried in vacuo to remove the solvent and oxalylchloride. The resultant solid was redissolved in 7 mL of DCM, cooled to−78° C., and 1.5 mL of pyridine was added. Then, 0.696 g of2-bromo-4,5-dimethoxy aniline (3 mmol) was added in 5 mL of DCM. A solidformed causing stirring to be difficult, and therefore, an additional 6mL portion of DCM was added. The mixture was warmed to 23° C., and afterthe reaction was complete, quenched with 20 mL of water. The layers wereseparated, the organic was washed with 20 mL of brine, dried withNa₂SO₄, and concentrated. The crude product was purified by columnchromatography and then PTLC (0.5% MeOH in DCM as eluent) to give 1.05 g(88% yield) of benzamide (2) as an off-white solid. ¹H NMR (200 MHz,CDCl₃) δ 8.25 (bs, 2H, overlapping peak), 7.57 (d, J=2 Hz, 1H), 7.48(dd, J=2 Hz, 8.4 Hz, 1H), 7.06 (s, 1H), 6.98 (d, J=8.4 Hz, 1H),4.09-3.99 (3 overlapping singlets, 9H), 3.90 (s, 3H).

To 0.049 g (0.14 mmol) of 2 in 3 mL of DCM was added 2.5 mL 1 M BBr₃ inDCM. The solution was stirred 18 h. The mixture was quenched with 10 mLMeOH and concentrated. The concentrate was diluted again with 5 mL ofMeOH, and 20 mL of MeOH was added. The resultant solid was filtered,collected, and dried under vacuum overnight to give 0.012 g (29% yield)of SA-52 as a brown solid. ¹H NMR (200 MHz, CDCl₃) 8 HRMS calculated forC₁₃H₁₁BrNO₅ (M+H)⁺ 339.9821. found 339.9828

Example 2 Synthesis of SA-53

To 0.401 g (1.5 mmol) of 2-bromo-4,5-dimethoxybenzoic acid (3) in 4 mLof DCM was added 0.4 mL (4.7 mmol) of oxalyl chloride, and 1 drop ofDMF. The mixture was stirred for 7 h, concentrated, dried in vacuo in asimilar manner to compound 2, and diluted with 5 mL of DCM. Thissolution was cooled to −78° C., treated with 0.8 mL pyridine and 0.137(1 mmol) 3,4-methylenedioxy aniline. The mixture was then brought to 23°C., stirred 16 h, quenched with 10 mL of water, and the resultant layersseparated. The organic layer was washed twice with 10 mL of water, driedwith Na₂SO₄, and concentrated. The crude product was purified by PTLCusing 10% EtOAc in DCM as the eluent to give 0.310 g (82% yield) ofbenzamide 4 as a brown solid. ¹H NMR (200 MHz, CDCl₃) δ 7.90 (bs, 1H),7.40 (bs, 1H), 7.27 (d, J=2.6 Hz, 1H), 7.15 (s, 1H), 6.96 (d, J=8.6 Hz,1H), 6.81 (d, J=8.6 Hz, 1H), 6.00 (s, 2H), 3.93 (s, 6H), HRMS calculatedfor C₁₆H₁₅BrNO₅ (M+H)⁺ 380.0134. found 380.0145.

Example 3 Synthesis of SA-54

To 0.230 g (0.58 mmol) of benzamide 2 was added 0.017 g of CuI (0.09mmol), 0.031 g (0.17 mmol) of 1,10 phenanthroline, and 0.560 g (1.7mmol) of Cs₂CO₃. The mixture was suspended in 5 mL of diglyme, andheated to 140° C. for 20 h. The mixture was diluted with 30 mL DCM,washed three times with 20 mL of water, dried (Na₂SO₄), and concentratedto give an orange oil as the crude product. This was heated under vacuum(1 mm Hg) until a light orange solid formed to give 0.135 g (75% yield)of benzoxazole 5, which was used without further purification. ¹H NMR(200 MHz, CDCl₃) δ 7.74 (dd, J=2.0 Hz, 8.4 Hz, 1H), 7.67 (d, J=2.2 Hz,1H), 7.21 (s, 3H), 7.10 (s, 3H), 6.95 (d, J=8.4 Hz, 1H), 3.98-3.89 (4overlapping singlets, 12H).

To 0.071 g (0.23 mmol) of 5 in 5 mL of DCM at 0° C. was added 2 mL 1MBBr₃ in DCM. The dark brown mixture was stirred 6 h, quenched with 5 mLMeOH, and concentrated. The MeOH dilution-concentration procedure wasrepeated three more times to give 0.081 g of crude product. This productwas purified by PTLC using 5% MeOH/DCM followed by 10% MeOH/DCM as theeluent and gave 0.023 g (39% yield) of SA-54 as an off-white solid. ¹HNMR (200 MHz) δ□8.84 (bs, 2H), 8.34 (bs, 1H), 7.35 (bs, 1H), 7.47 (d,J=2 Hz, 1H), 7.40 (dd, J=2 Hz, 8.2 Hz, 1H), 7.05 (s, 2H), 7.01 (s, 2H).HRMS Calculated for C₁₃H₁₀NO₅ (M+H)⁺ 260.0586 found 260.0559.

Example 4 Synthesis of SA-55

To 10.00 g (51 mmol) of methyl 3,4 dimethoxybenzoate (6) in 50 mL ofAcOH at 0° C. was added 8.90 g (56 mmol) of Br₂ in 50 mL of AcOH over1.5 h. The ice bath was removed and the mixture stirred 45 min. Thereaction was quenched by pouring into 700 mL of H₂O, stirred 30 min,left quiescent for 1 h, and filtered. The collected solid was washedwith H₂O and washed with sat. aq. Na₂S₂O₃. The solid was partiallydried, dissolved in 300 mL hot MeOH, and the resultant solution wascooled. The cool methanolic solution of product was treated with 200 mLof H₂O and the white solid filtered to give 8.92 g (64% yield) ofmethyl-2-bromo-4,5-dimethoxybenzoate (7) as a white powder. The compoundmatched the physical and spectral properties of the known compound.

A mixture of 0.960 g (3.48 mmol) of 7, 1.60 g (35 mmol) of hydrazinehydrate (62% hydrazine), and 5 mL of EtOH was refluxed for 15 h. Themixture was cooled to −20° C., vacuum filtered, washed with 50 mL ofice-cold 1:1 EtOH:H₂O, and dried to give 0.832 g (87% yield) of(2-bromo-4,5-dimethoxy benzoyloxy)hydrazine (8) as a white, needle-likecrystalline solid. The above procedure was repeated on 2.79 g of thestarting ester 7 to result in 2.77 g (99% yield) of 8. ¹H NMR (200 MHz,CDCl₃) δ 6.97 (s, 1H), 6.86 (s, 1H), 3.85 (s, 6H). HRMS Calculated forC₉H₁₂O₃N₂Br 275.0031. found 275.0037.

To 2.15 g (11.8 mmol) of 3,4-dimethoxybenzoic acid (1) in 10 mL of DCMwas added sequentially 2.5 mL (29.1 mmol) oxalyl chloride and 0.2 mL ofDMF. The mixture was stirred for 16 h during which time it became aclear, light yellow solution. This solution was concentrated and driedthoroughly to remove the excess oxalyl chloride to generate the crudeacid chloride as a light yellow solid. This solid was taken up in 20 mLof DCM, the solution cooled to 0° C., and treated with 10 mL of pyridineand 0.25 g of DMAP. The resultant solution was treated with 1.69 g (6.15mmol) of 8 in 10 mL DCM and 10 mL of pyridine. The mixture was stirred 3h at 0° C. and warmed to 23° C. The reaction was stirred an additional16 h, concentrated, taken up in 50 mL of EtOAc, and the layersseparated. The aqueous was extracted once more with 50 mL EtOAc. Thecombined organic layers were washed three times with 100 mL H₂O, dried(Na₂SO₄), and concentrated. The concentrate was purified by Flash 40+Mcolumn chromatography (Biotage) eluting first with 150 mL of 1:1EtOAc/Hex and then 2 L 5:1 EtOAc/Hex to give 1.27 g (47% yield) ofhydrazide 9 as a yellow-brown powder. The reaction was repeated using2.20 g of 3,4 dihydroxybenzoic acid (1) and 2.77 g of hydrazine 8 togive 2.60 g (49% yield) of hydrazide 9. NMR (200 MHz, CDCl₃) δ 9.91 (d,J=5.2 Hz, 1H), 9.53 (d, J=5.0 Hz, 1H), 7.46 (dd, J=2.0 Hz, 8.4 Hz, 1H),7.41 (d, J=2.2 Hz, 1H), 7.23 (s, 1H), 6.98 (s, 1H), 6.80 (d, J=8.4 Hz,1H), 3.96-3.89 (4 overlapping singlets, 12H).

A solution of 0.352 g of intermediate 9 in 3 mL of POCl₃ was refluxedfor 3 h. The reaction mixture is cooled, poured into 125 mL of water,and sonicated for one minute. The suspension was allowed to stand for 1h, and the solid was filtered, washed with excess water, collected, andair-dried to give 0.302 g (90% yield) of brominatedtetramethoxyoxadiazole (10) as a white solid. ¹H NMR (200 MHz, CDCl₃) δ7.71 (dd, J=2.0 Hz, 8.4 Hz, 1H), 7.67 (d, J=2.0 Hz, 1H), 7.57 (s, 1H),7.17 (s, 1H), 6.98 (d, J=8.2 Hz, 1H), 3.96 (4 overlapping singlets,12H). HRMS Calculated for C₁₄H₁₀BrN₂O₅ (M+H)⁺ 421.0339. found 421.0334.

A mixture of 0.038 g of intermediate 10 in 3 mL of DCM was cooled to−78° C., and treated dropwise with a solution of 0.450 g of BBr₃ in 5 mLof DCM. The mixture was stirred at −78° C. for 1 h, then at 23° C. for2.5 h. The mixture was quenched by adding it carefully to 5 mL of MeOHin a 100 mL flask. The methanol solution was concentrated to 1 mL,diluted with 5 mL water, and filtered to give 0.017 g (56% yield) ofSA-55 as a light yellow solid. ¹H NMR (200 MHz, CDCl₃) δ 10.5-9.5(overlapping broad singlets, 4H), 7.56 (d, J=2 Hz, 1H), 7.52 (s, 1H),7.48 (dd, J=2 Hz, 8.6 Hz, 1H), 7.25 (s, 1H), 7.04 (d, J=8.4 Hz, 1H)

Example 5 Synthesis of SA-57

To a thick-walled 15 mL tube with a resealable Teflon screw-cap wasadded 0.584 g (4.32 mmol) of 3,4-dihydroxybenzonitrile (12), 5 mL oftriethylene glycol, 1.172 g of NaSH.xH₂O, and 0.25 mL of concentratedH₂SO₄. The tube was sealed with the cap, and the mixture was warmed to110° C. and stirred for 3 days at this temperature. The reaction wasquenched by pouring into 100 mL sat. aq. NH₄ ⁺Cl, and extracted twicewith 50 mL of EtOAc. The combined organics were washed three times with15 mL water, dried with NaSO₄, and concentrated to give 0.512 g (71%yield) of 13 as a golden colored solid. ¹H NMR (200 MHz, CDCl₃) δ 9.67(s, 1H), 9.29 (s, 1H), 7.56 (dd, J=1.8 Hz, 8.2 Hz), 7.49 (d, J=1.8 Hz),6.94 (d, J=8.2 Hz), 6.09 (s, 2H). ¹³C NMR (50 MHz, CDCl₃) δ 198.9,150.5, 147.4, 133.7, 123.3, 108.2, 107.8, 102.3.

A solution of 0.40 g (2.37 mmol) of 13 in 7 mL of DMSO was treated with12 drops of concentrated HCl, warmed to 38° C. for 18 h, and poured into25 mL of brine. The resultant solid was filtered and washed with waterto give 0.21 g (59% yield) of SA-57 as a yellow solid. ¹H-NMR (DMSO-d6)9.85 (bs, 1H) 9.52 (bs, 1H), 9.43 (bs, 1H), 9.28 (bs, 1H), 7.69 (d, 1H,J=2 Hz), 7.59 (dd, 1H, J=2.2, 8.2 Hz), 7.46 (d, 1H, J=2 Hz), 7.38 (dd,1H, J=2.2, 8.2 Hz), 6.90 (d, 1H, J=8.2 Hz), 6.86 (d, 1H, J=8.4 Hz).¹³C-NMR (DMSO-d6) 187.8, 173.4, 150.2, 148.5, 146.4, 145.9, 124.6,122.0, 120.4, 120.2, 116.8, 116.2, 115.7, 114.5. HRMS-ESI Calculated forC₁₄H₁₁N₂O₄S (M+H)⁺ 303.0440. found 303.0448.

Example 6 Synthesis of SA-58

To a solution of 2,5-dibromothiophene (14) (242 mg, 1 mmol),(3,4-dimethoxy phenyl)boronic acid (15) (455 mg, 2.5 mmol), andPd(PPh₃)₄ (58 mg, 0.05 mmol) in dioxane (10 mL) was added Na₂CO₃ (12 mL,2.0 M aqueous solution). The resultant mixture was purged with nitrogenand stirred rapidly while heating at 90° C. overnight. The reactionmixture was cooled to 23° C., acidified with 1M HCl and extracted withEtOAc. The combined organic extracts were washed with H₂O, dried overMgSO₄, filtered and concentrated under reduced pressure.2,5-bis(3′,4′-dimethoxyphenyl)thiophene (16) was obtained quantitativelyas a green-yellow solid after the purification by column chromatography(10%-20% EtOAc in hexanes).

To a solution of 2,5-bis(3′,4′-dimethoxyphenyl)thiophene 16 (110 mg, 0.3mmol) in dry dichloromethane at −78° C. was added BBr₃ (3 mL, 1Msolution in DCM, 2.5 equiv per methoxy function) dropwise. The reactionmixture was stirred at −78° C. for 3 h, warmed to 23° C., and stirred 16h under nitrogen atmosphere. Water (10 mL) was added to quench thereaction, and the aqueous layer was extracted with EtOAc. The combinedorganic layer was washed with brine, dried over MgSO₄, filtered, andconcentrated under reduced pressure. The product was purified byrecrystallization in MeOH/DCM and SA-58 was obtained quantitatively as agreenish solid.

Example 7 Synthesis of SA-59, SA-60 and SA-61

3-bromo-2,5-bis(3′,4′-dimethoxyphenyl)thiophene (18) was prepared by thereaction of 2,3,5-tribromothiophene (17) (321 mg, 1 mmol) and(3,4-dimethoxyphenyl)boronic acid (15) (419 mg, 2.3 mmol) according tothe similar procedure for compound 16. The reaction mixture was purifiedby column chromatography (10%-30% EtOAc in hexanes) and afforded 18 (337mg, 77% yield) as a yellow solid. SA-60 was also isolated (97 mg, 20%yield) from the reaction above as a dark yellow solid.

SA-59 was prepared by the reaction of3-bromo-2,5-bis(3′,4′-dimethoxyphenyl)thiophene (18) (258 mg, 0.59 mmol)and BBr₃ (6 mL, 6 mmol) according to the similar procedure for compoundSA-58. SA-59 (157 mg, 70% yield) was obtained after preparative thinlayer chromatography (PTLC) purification (10% MeOH in DCM) as a greensolid.

SA-61 was prepared by the reaction of2,3,5-tri(3′,4′-dimethoxyphenyl)thiophene (SA-60) (68 mg, 0.14 mmol) andBBr₃ (2 mL, 2 mmol) according to the similar procedure for compoundSA-58. SA-61 (34 mg, 60% yield) was obtained after PTLC purification(10% MeOH in DCM) as a brown oil.

Example 8 Synthesis of SA-62

Compound 19 was prepared by the reaction of 2,5-dibromothiophene (17)(1.14 g, 5.24 mmol) and (3,4-dimethoxyphenyl) boronic acid (15) (910 mg,5 mmol) according to the similar procedure for compound 16. The reactionmixture was purified by flash column chromatography (FCC) (5%-20% EtOAcin hexanes) and afforded compound 19 (509 mg, 34% yield) as a yellowishcrystal. Compound 16 was also isolated (578 mg, 65% yield) as a yellowsolid.

SA-62 was prepared by the reaction of2-bromo-5-(3,4-dimethoxyphenyl)thiophene (19) (60 mg, 0.2 mmol) and BBr₃(1M in DCM, 1 mL, 1 mmol) according to the similar procedure forcompound SA-52 and isolated as a green solid in quantitative yield.

Example 9 Synthesis of SA-63

Compound 21 was prepared by the reaction of2-bromo-5-(3,4-dimethoxyphenyl)thiophene (19) (449 mg, 1.5 mmol) and(3,4,5-trimethoxyphenyl)boronic acid (20) (382 mg, 1.8 mmol) accordingto the similar procedure for compound 16 and purified by columnchromatography (10%-20% EtOAc in hexanes), then recrystallization(EtOAc) provided the desired compound as a yellow solid (524 mg, 91%yield).

SA-63 was prepared by the reaction of2-(3,4-dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)thiophene (21) (47 mg,0.12 mmol) and BBr₃ (1M in DCM, 0.8 mL, 0.8 mmol) according to thesimilar procedure for compound SA-52. SA-63 was obtained quantitativelyas dark blue solid.

Example 10 Synthesis of SA-64

To a solution of dioxane/EtOH/H₂O (6 mL, 1/1/1) in a microwave reactionvial (Biotage) was added 2,5-dibromothiazole (22) (122 mg, 0.5 mmol),(3,4-dimethoxyphenyl)boronic acid (15) (218 mg, 1.2 mmol), Pd(PPh₃)₄ (29mg, 0.025 mmol) and Cs₂CO₃ (0.72 g, 2.2 mmol). The mixture was purgedwith nitrogen and heated in a microwave reactor (Biotage) to 160° C. for1 h. The reaction mixture was cooled to 23° C., acidified with 1M HCluntil the pH was 1, and extracted with EtOAc. The combined organicextracts were washed with H₂O before being dried over MgSO₄, filtered,and concentrated under reduced pressure. Compound 24 was purified bycolumn chromatography (5%-35% EtOAc in hexanes) as brown-yellow solid(28 mg, 16% yield). 5-bromo-2-(3,4-dimethoxyphenyl)thiazole (23) wasalso isolated from the reaction above as brown-yellow crystalline solid(28 mg, 19% yield).

SA-64 was prepared by the reaction of2,5-bis(3,4-dimethoxyphenyl)thiazole (24) (28 mg, 0.078 mmol) and BBr₃(1M in DCM, 0.75 mL, 0.75 mmol) according to the similar procedure forcompound SA-58, and was obtained as a yellow solid (14 mg, 60% yield).

Example 11 Synthesis of SA-65

A mixture of 0.100 g (0.237 mmol) of 10, 0.600 g (1.03 mmol) ofhexabutylditin, 5 mL of PhMe, and 1 mL of TEA was degassed by nitrogenpurge. Then 0.050 g of Pd(PPh₃)₄ was added. The reaction mixture wasrefluxed for 16 h. At this time, the reaction was incomplete, but adecomposition product was seen in addition to the desired product. Thereaction was at this point concentrated and purified by PTLC to preventfurther decomposition to give 0.090 g (60% yield) of SA-65 as anoff-white solid. NMR (200 MHz, CDCl₃) δ 7.68-7.65 (overlapping peakdoublet and doublet of doublets, 2H), 7.53 (s, 1H), 7.16 (s, 1H), 6.98(d, J=9.0 Hz, 1H), 3.99-3.96 (4 overlapping singlets, 12H), 1.55 (m,6H), 1.44 (m, 6H), 1.15 (m, 6H), 0.84 (t, J=7.2 Hz, 9H).

Example 12 Synthesis of SA-66

A mixture of 0.174 g of Lawesson's reagent, 0.175 g of compound 9, and20 mL of PhMe was heated to 60° C. for 3 h. The mixture wasconcentrated, and applied directly to a PTLC plate for purification. Thebrominated thiadiazole 11 was purified by PTLC and the middle of thedesired product spot (fluoresces blue under UV light) was collected togive 0.112 g (65% yield) of compound 11 as an off-white to brown solid.

The above experiment was repeated using 1.13 g of 9, 1.21 g ofLawesson's reagent, and 200 mL of PhMe. The mixture was refluxed 3 h,and quenched with 150 mL of water. The layers were separated, and theaqueous extracted twice with 30 mL of EtOAc. The organic layers werecombined and washed twice with 50 mL 1 N aq. HCl, twice with 50 mLsaturated aq. NaHCO₃, once with 25 mL of water, and once with 50 mL ofbrine. The organic was dried, concentrated, and purified by PTLC to give1.04 g of the desired 11 as a yellow solid (97% yield). ¹H NMR (200 MHz,CDCl₃) δ 7.88 (s, 1H), 7.68 (bs, 1H), 7.51 (d, J=7.8 Hz, 1H), 7.15 (s,1H), 6.95 (d, J=8.4 Hz, 1H), 4.00-3.95 (4 overlapping singlets, 12H).

A solution of 0.051 g of 11 in 5 mL DCM was cooled to −78° C., treatedwith 1.5 mL of 2 M BBr₃ in DCM, stirred at −78° C. for 0.5 h, andstirred at 23° C. for 3 h. The mixture was quenched with watercarefully, poured into 100 mL brine, and extracted twice with 75 mL ofEtOAc. The combined organic layers were dried and concentrated to yield36 mg (81% yield) of the crude title compound as a yellow solid. Themixture was recrystallized from hot MeOH and precipitated with water togive 0.021 g (50% yield) of SA-66 as a brown solid.

The above experiment was repeated on 0.077 g of starting material usingthe same procedure with the exception of slightly different stirringtimes (1 h at −78° C., 4 h at 23° C.) to give 0.054 g (83% yield) ofSA-66. ¹H NMR (200 MHz, DMSO-d₆) δ 10.10 (s, 1H), 9.78 (s, 1H), 9.68 (s,1H), 9.47 (s, 1H), 7.55 (s, 1H), 7.43 (d, J=1.8 Hz, 1H), 7.29 (dd, J=1.8Hz, 8.0 Hz, 1H), 7.13 (s, 1H), 6.87 (d, J=8.2 Hz, 1H).

Example 13 Synthesis of SA-67

To 0.285 g (1.57 mmol) of 3,4dimethoxybenzoic acid (1) in 5 mL of DCMwas added 0.3 mL (3.5 mmol) of oxalyl chloride and one drop of DMF. Themixture was stirred for 2 h, quenched with hydrazine hydrate (3 mL),concentrated, and dried. The solid was taken up in 25 mL water,sonicated five minutes, filtered, and the resultant solid washed with 40mL water. The solid was dried, taken up in 20:1 DCM:DMF, and purified byPTLC (8:2 EtOAc:Hexanes) to give 0.056 g (20% yield) of 25 as anoff-white solid. ¹H NMR (200 MHz, DMSO-d₆) δ 10.30 (s, 2H), 7.58 (d,J=8.4 Hz, 1H), 7.51 (bs, 1H), 7.18 (d, J=8.4 Hz, 1H), 3.82 (bs, 12H).

To 0.050 g (0.14 mmol) of hydrazide 25 was added 3 mL of POCl₃. Themixture was refluxed 2 h, quenched with 50 g of ice, allowed to warm to23° C., and extracted twice with 25 mL of EtOAc. The organic layers werewashed once with 25 mL water, twice with 25 mL of brine, dried withNa₂SO₄, and concentrated to give 0.043 g (93% yield) of 26 as a whitesolid. ¹H NMR (200 MHz, CDCl₃) δ 7.72 (d, J=8.0 Hz, 2H), 7.60 (d, J=1.2Hz, 1H), 7.07 (d, J=8.4 Hz, 1H), 3.89 (s, 6H), 3.87 (s, 6H).

To 0.038 g (0.11 mmol) of oxadiazole 26 was added 3 mL of DCM. Thesolution was cooled to −78°, and 5 mL of DCM containing 0.450 g (1.8mmol) of BBr₃ was added. The mixture was stirred at −78° C. for 1 h, at23° C. for 2.5 h, quenched by pouring into 5 mL of MeOH, andconcentrated to 1 mL. The resultant oil was diluted with 5 mL water andthe resultant precipitate was filtered to give 0.017 g (56% yield) ofSA-67 as a yellow solid. ¹H NMR (200 MHz, DMSO-d₆) δ 9.74 (bs, 2H), 9.51(bs, 2H), 7.43 (d, J=2.0 Hz, 1H), 7.37 (dd, J=2.0 Hz, 8.4 Hz, 1H), 6.93(d, J=8.0 Hz, 1H).

Example 14 Synthesis of SA-68

4-azido-1,2-dimethoxybenzene (27) (62 mg, 0.31 mmol),4-ethynyl-1,2-dimethoxy benzene (28) (52 mg, 0.31 mmol), sodiumascorbate (0.25 mL of 1M solution, 0.25 mmol), CuSO₄ (0.020 mL of 1Msolution, 0.020 mmol) and tBuOH/H₂O (2 mL, 1/1) were added to a vial.The reaction mixture was purged with nitrogen, stirred at 23° C.overnight, poured into water at 0° C., and the resultant brown solid wasfiltered. This solid was washed with water (1 mL) and Et₂O (1 mL).Compound 29 (99 mg, 93% yield) was used in the next step without furtherpurification.

SA-68 was prepared by the reaction of1,4-bis(3,4-dimethoxyphenyl)-1H-1,2,3-triazole (29) (67 mg, 0.20 mmol)and BBr₃ (1M in DCM, 0.79 mL, 0.79 mmol) according to the similarprocedure for compound SA-52. SA-68 was obtained as a dark brown solid(56 mg, 98% yield).

Example 15 Synthesis of SA-69

To a solution of dioxane/EtOH/H₂O (6 mL, 1/1/1) were added2,5-dibromothiazole (22) (756 mg, 3.1 mmol),(3,4-dimethoxyphenyl)boronic acid (15) (380 mg, 2.1 mmol), Pd(PPh₃)₄ (58mg, 0.05 mmol) and Cs₂CO₃ (1.4 g, 4.4 mmol) in a 20 mL microwavereaction vial (Biotage). The solution was purged with nitrogen andheated in a microwave reactor (Biotage) to 160° C. for 30 min. Thereaction mixture was cooled to 23° C., acidified with 1M HCl until pH=1,and extracted with EtOAc. The combined organic extracts were washed withH₂O before being dried over MgSO₄, filtered, and concentrated underreduced pressure. Compound 23 was purified by flash columnchromatography (8%-50% EtOAc in hexanes) as brown-yellow solid (193 mg,32% yield). 2,5-bis(3,4-dimethoxyphenyl)thiazole (24), was also isolatedas a brown-yellow solid (34 mg, 5% yield).

SA-69 was prepared by the reaction of5-bromo-2-(3′,4′-dimethoxyphenyl)thiazole (23) (78 mg, 0.26 mmol) andBBr₃ (1M in DCM, 0.65 mL, 0.65 mmol) according to the similar procedurefor compound SA-52, and was obtained as a brown-yellow solidquantitatively.

Example 16 Synthesis of SA-70

A mixture of 0.996 g (6 mmol) 3,4-dimethoxybenzaldehyde (30), 1.25 g ofTosMIC (6.4 mmol), and 0.923 g (6.6 mmol) of K₂CO₃ was refluxed in 30 mLof MeOH for 3 h. The reaction mixture was quenched by pouring into 200mL of a 1:1 mixture of brine and water, cooled to −20° C. for 1 h, andthe resultant solid filtered to give 0.952 g (77% yield) of 31 as anoff-white solid. ¹H NMR (200 MHz, CDCl₃) δ 7.85 (s, 1H), 7.22 (s, 1H),7.20 (overlapping peak, 1H), 7.15 (dd, J=2.0 Hz, 12.4 Hz, 1H), 6.88 (d,J=1H), 3.91 (s, 3H), 3.88 (s, 3H).

A suspension of 0.212 g of Na₂CO₃ (2 mmol), 0.262 g of PPh₃ (1 mmol),0.206 g (1 mmol) of 31 and 0.316 g of 4-iodoveratrole (1.2 mmol) wasformed in 1 mL of DMF. Then, 0.190 g (1 mmol) of CuI was added. Themixture was stirred at 160° C. for 3 h and quenched by pouring into 50mL of water containing 5% NH₄ ⁺OH⁻. The product was extracted with 50 mLDCM, the organic layer dried and concentrated, and the crude productpurified by PTLC to give 0.174 g (51% yield) of 32 as a yellow solid. ¹HNMR (200 MHz, CDCl₃) δ 7.60 (d, J=8.4 Hz, 1H), 7.54, (bs, 1H), 7.24 (s,1H), 7.19 (bs, 1H), 7.10 (s, 1H), 6.90-6.83 (m, 2H), 3.91-3.86 (4overlapping multiplets, 12H). ¹³C NMR (50 MHz, CDCl₃) δ 160.7, 150.9,149.3, 149.2, 132.1, 132.0, 122.0, 121.2, 120.4, 119.3, 117.1, 111.5,111.1, 109.1, 107.5, 56.01, 55.92.

A mixture of 0.075 g (0.22 mmol) of 32 in 15 mL DCM was treated with0.500 g (2 mmol) of BBr₃ at −78° C. The mixture was stirred at −78° C.for 0.5 h, then 2 h at 23° C. The reaction was then quenched with 5 mLMeOH, concentrated to 1 mmol and diluted with 5 mL of water. Theresultant precipitate was filtered to give 0.021 g (33% yield) of SA-70as a yellow solid. ¹H NMR (200 MHz, DMSO-d₆) δ 7.40 (s, 2H), 7.31 (dd,J=2.0 Hz, 8.0 Hz, 1H), 7.13 (d, J=2.2 Hz), 7.05 (dd, J=2.2 Hz, 8.2 Hz,1H), 6.86 (d, J=7.8 Hz, 1H), 6.82 (d, J=7.8 Hz, 1H). HRMS Calculated forC₁₅H₁₂NO₅ (M+H)⁺ 286.0715. found 286.0717.

Example 17 Synthesis of SA-72

A solution of 0.115 g (0.32 mmol) of 25 in 30 mL of PhMe was treatedwith 0.140 g of Lawesson's reagent (0.35 mmol) and stirred 3 h at 100°C. The reaction mixture was poured into 75 mL water, shaken vigorously,and extracted with 50 mL EtOAc. The organic layers were combined, washedwith 25 ml saturated aqueous NaHCO₃, washed with 50 mL brine, dried, andconcentrated. The crude product was purified by PTLC to give 0.088 g(74% yield) of 33 as a yellow solid. ¹H NMR (200 MHz, CDCl₃) δ 7.60 (bs,2H), 7.37 (d, J=8.2 Hz, 2H), 6.86 (d, J=8.4 Hz, 2H), 3.95 (s, 6H), 3.90(s, 6H). ¹³C NMR (200 MHz, CDCl₃) 167.3, 151.6, 149.4, 123.2, 121.6,111.2, 110.0, 56.1, 56.0. HRMS Calculated for C₁₆H₁₉N₂O₄S (M+H)⁺359.1066. found 359.1061.

A solution of 0.077 g (0.22 mmol) of 33 and 3 mL DCM was treated with0.250 g BBr₃ at −78° C. The reaction mixture was stirred 1 h, treatedwith 0.250 g BBr₃, and stirred an addition 15 min at −78° C. The mixturewas warmed to 23° C., stirred for 1 h, quenched with 10 mL MeOH, andconcentrated. The concentrate was taken up in 1 mL MeOH, warmed until asolution, and precipitated with 10 mL water. The resultant solid wasfiltered and dried to give 0.054 g of SA-72 (83% yield) as an off-whitesolid. ¹H NMR (200 MHz, DMSO-d₆) 10.0-9.0 (broad peak, 4H), 7.41 (d,J=2.2 Hz, 2H), 7.24 (dd, J=2.2 Hz, 8.2 Hz, 1H), 6.87 (d, J=8.2 Hz, 2H).

Example 18 Synthesis of SA-74

To a mixture of 0.219 g (0.5 mmol) of 11 in 20 mL PhMe was added 1.09 g(Bu₃Sn)₂ and 2 mL, of triethylamine. The mixture was degassed via anitrogen purge, treated with 0.150 g (0.13 mmol) of Pd(PPh₃)₄, andrefluxed for 15 h. The mixture was concentrated and purified directly byPTLC by eluting the plates with 1% TEA in hexanes then 50% EtOAc inhexanes with 1% TEA successively. This gave 0.224 g (69% yield) SA-74 asan off-white solid.

Example 19 Synthesis of SA-75 and SA-76

A solution of 0.051 g of SA-74 in 3 mL of DCM was treated with a 1 Miodine in DCM solution until the orange/yellow color of the iodinepersists. The mixture was quenched with a 1 M solution of KF in MeOH (2mL) then sat. aq. Na₂S₂O₃ (2 mL). The mixture was extracted twice with10 mL EtOAc. The organic layers were combined, washed with 10 mL water,dried and concentrated to give the crude title compound. The crudeproduct was purified by PTLC with another run of this reaction using0.082 g of SA-74 and run in the same manner as described above. Thisgave 0.012 g (12% yield) of SA-75 as a yellow solid. ¹H NMR (200 MHz,CDCl₃) δ 7.73 (d, J=2.2 Hz, 1H), 7.59 (s, 1H), 7.54 (dd, J=2.2 Hz, 8.4Hz), 7.42 (s, 1H), 6.98 (d, J=8.4 Hz, 1H), 4.03-3.95 (4 overlappingsinglets, 12H).

A solution of 0.008 g of SA-75 in 1 mL of DCM was cooled to 0° C.,treated with 0.5 mL of 1 M BBr₃ in DCM, and stirred for 1 h. Thereaction was treated with an additional 1 mL portion of 1 M BBr₃ in DCM,stirred an additional 2 h at 0° C., and warmed to 23° C. The reactionmixture was stirred an additional 0.5 h, quenched with 3 mL MeOH, andconcentrated to 0.5 mL. The product was precipitated with 5 mL water,and the resultant yellow solid was filtered and dried to give (0.002 g)of SA-76 as a yellow-green solid. ¹H NMR (200 MHz, DMSO-d₆) δ 9.93-9.50(overlapping broad singlets, 4H), 7.42-7.28 (overlapping irresolvablepeaks, 4H), 6.98 (d, J=8.2 Hz, 1H).

Example 20 Synthesis of SA-77 and SA-78

To a round-bottom flask charged with5-(5-(3,4-dihydroxyphenyl)thiophen-2-yl)benzene-1,2,3-triol (SA-63) (145mg, 0.46 mmol), 4-methylbenzenesulfonic acid (p-TsOH.H₂O) (8.7 mg, 0.046mmol), and 2,2-dimethoxypropane (0.45 mL, 3.7 mmol) were added acetone(3 mL) and benzene (15 mL). A short column with 4 Å molecular sieves anda condenser were installed on the flask. The reaction was refluxed for15 h. An additional portion of 2,2-dimethoxypropane (0.45 mL, 3.7 mmol)was added, and the reaction was refluxed for another 24 h. The solutionwas concentrated at reduced pressure and purified by PTLC, whichafforded 34 as a white solid (53 mg, 29%).

To a solution of6-(5-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)thiophen-2-yl)-2,2-dimethylbenzo[d][1,3]dioxol-4-ol(34) (53 mg, 0.13 mmol), triethylamine (0.056 mL, 0.40 mmol) and DCM (2mL) was added trifluoromethanesulfonic anhydride (0.034 mL, 0.20 mmol)at 0° C. The reaction was warmed to 23° C. after 30 min and extractedwith DCM. The organic layer was washed with sat. aq. NaHCO₃, H₂O andbrine. The organic layers were dried over MgSO₄, concentrated, and 35was obtained as yellowish oil (53 mg, 75% yield).

To6-(5-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)thiophen-2-yl)-2,2-dimethylbenzo[d][1,3]dioxol-4-yltrifluoromethanesulfonate (35) (32 mg, 0.061 mmol) was added Pd(PPh₃)₄(14 mg, 0.012 mmol), LiCl (26 mg, 0.61 mmol), bistributylditin (0.153mL, 0.305 mmol) and dioxane (2 mL) (plus Triethylhylamine??). Thesolution was purged with nitrogen and refluxed for 3.5 h. The reactionwas concentrated, evaporated and subjected to PTLC (8% EtOAc inhexanes). SA-77 was obtained as a yellowish oil (26 mg, 64% yield).

To the solution oftributyl(6-(5-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)thiophen-2-yl)-2,2-[d][1,3]dioxol-4-yl)stannane(SA-77) (26 mg, 0.039 mmol) and THF (1 mL) was added the solution of I₂(20 mg, 0.078 mmol) in THF (1 mL) dropwise. The reaction was stirred for10 min and concentrated, and 36 was obtained quantitatively as a yellowsolid after purification by PTLC (7% EtOAc in hexanes).

To6-(5-(2,2-dimethylbenzo[d][1,3]-dioxol-5-yl)thiophen-2-yl)-4-iodo-2,2-dimethylbenzo[d][1,3]-dioxole(36) (15 mg, 0.029 mmol) were added a few drops of H₂O andtrifluoroacetic acid (0.45 mL). The reaction was stirred at 23° C. for 1h, and afforded SA-78 quantitatively as a white crystal afterconcentration at reduced pressure.

Example 21 Synthesis of SA-79 and 80

To 4-(5-bromothiazol-2-yl)benzene-1,2-diol (SA-69) (90 mg, 0.32 mmol)was added 4-dimethylaminopyridine (DMAP) (117 mg) and acetic anhydride(3 mL). The reaction was stirred at 23° C. for 1 h before it wasextracted with EtOAc. The organic layer was washed with sat. aq. NaHCO₃,H₂O and brine and dried over MgSO₄. SA-79 was obtained as yellow solid(74 mg, 65% yield) after PTLC (20% EtOAc in hexanes).

To a solution of 4-(5-bromothiazol-2-yl)-1,2-phenylene diacetate (SA-79)(36 mg, 0.10 mmol), Pd(PPh₃)₄ and dioxane (2 mL) was addedbistributyltin (0.25 mL, 0.50 mmol). The solution was purged withnitrogen and refluxed for 1.5 h. PTLC (25% EtOAc in hexanes) providedSA-80 as yellow oil (22 mg, 39% yield).

Example 22 Synthesis of SA-81

To a solution of nBuLi (0.34 mL, 2.5 M in hexanes) and THF (6 mL) wasadded 5-bromo-2-(3,4-dimethoxyphenyl)thiazole (23) (54 mg, 0.18 mmol)and additional THF (3 mL) was added dropwise at −78° C. under nitrogenatmosphere. SnBu₃Cl (0.15 mL) was added after the reaction was stirredat −78° C. for 30 min. After another 30 min, saturated aqueous NaHCO₃was added to quench the reaction. The mixture was extracted with EtOAcand the organic layer was washed with water and brine. PTLC (25% EtOAcin hexanes) provided SA-81 as a yellow oil (58 mg, 63% yield).

Example 23 Synthesis of SA-82

A mixture of 0.26 g (1 mmol) of 2-bromo-4,5-dimethoxybenzoic acid (3) in5 mL of DCM was treated with 0.3 mL of (COCl)₂ and two drops of DMF. Themixture was stirred for an additional hour after a yellow solution hadformed (about 2 h total). The mixture was concentrated and dried invacuo thoroughly, taken up in 15 mL of DCM, cooled to 0° C., and treatedwith 2 mL pyridine. Then 0.79 g of known amine 37 was added. The mixturewas warmed to 23° C., stirred 3 days, and quenched with water. Theproduct was extracted with 10 mL of DCM, the combined organic was washedwith 10 mL water, dried, and concentrated to give the crude product thatwas purified by PTLC (50% EtOAc in hexanes) to give 0.070 g (16% yield)of compound 38 as a light brown solid. ¹H NMR (200 MHz, CDCl₃) δ 7.67(dd, J=2.0 Hz, 8.4 Hz, 1H), 7.56 (apparent broad singlet, overlappingpeaks, 2H), 7.04 (s, 1H), 6.95 (d, J=6.95 Hz, 1H), 4.94 (d, J=4.2 Hz,1H), 3.98 (s, 3H), 3.95 (s, 3H), 3.86 (bs, 6H). HRMS Calculated forC₁₉H₂₂O₆NBr (M+H)⁺ 438.0552. found 438.0556.

A mixture of 0.103 g (0.24 mmol) of amide 38 in 10 mL of THF was treatedwith 0.117 g (0.29 mmol) of Lawesson's reagent. The mixture was refluxedfor 2 h, cooled, and concentrated. The crude product was purifieddirectly by PTLC to give 0.048 g (45% yield) of compound 39 as a lightbrown solid. ¹H NMR (200 MHz, CDCl₃) δ 7.78 (apparent singlet, 1H),7.55-7.50 (overlapping singlet, dd, 2H), 7.46 (s, 1H), 7.13 (s, 1H),6.94 (d, J=8.8 Hz, 1 h), 3.97-3.89 (overlapping singlets, 12H).

A solution of 0.031 g 39 (0.07 mmol) in 4 mL of DCM was cooled to −78°C., and treated with 1.5 mL of 1 M BBr₃ in DCM. The mixture was stirredfor 1 h at −78° C., 1 h at 23° C., and quenched with 2 mL of MeOH. Themixture was concentrated to 1 mL, diluted with 10 mL water, sonicated,and the resultant solid filtered to give 0.009 g (35% yield) of SA-82 asan off-white solid.

Example 24 Synthesis of SA-83

To 0.012 g (0.03 mmol) of SA-76 in 0.5 mL pyridine was added 0.05 mL ofacetic anhydride. The mixture was stirred at 140° C. for 3 h, cooled,and poured into 10 mL of water with 0.5 g NH₄ ⁺Cl⁻, and the product wasextracted with 5 mL 10% MeOH in DCM. The extract was concentrated, andpurified by PTLC to give 0.005 g (8.3*10⁻³ mmol) of SA-83 as a brownoil. ¹H NMR (200 MHz, CDCl₃) δ 7.94-7.88 (3 overlapping irresolvablepeaks, 3H), 7.82 (s, 1H), 7.36 (d, J=9.0 Hz), 2.34-2.32 (4 overlappingsinglets, 12H).

Example 25 Synthesis of SA-84

A mixture of 0.058 g (0.132 mmol) of amide 38 was refluxed in 2 mLPOCl₃. The solution was added to 50 mL of water and sonicated toprecipitate a light yellow solid, which was filtered, washed with coldwater, and dried to give 0.041 g (74% yield) of 40 as a yellow solid. ¹HNMR (200 MHz, CDCl₃) δ 7.61 (s, 1H), 7.37 (d, J=2.0 Hz, 1H), 7.31 (dd,J=2.2 Hz, 8.4 Hz, 1H), 7.25 (s, 1H), 7.18 (s, 1H), 6.96 (d, J=8.4 Hz,1H), 3.96 (overlapping singlets, 12H). HRMS Calculated for C₁₉H₁₉BrNO₅(M+H)⁺ 420.0448. found 420.0465.

To 0.100 g (0.24 mmol) of 40 in 20 mL of DCM at −78° C. was added 2.5 mLof 1 M BBr₃ in DCM. The reaction was warmed to 23° C., stirred for 3 h,and quenched with 10 mL of MeOH. The reaction mixture was concentrated,and water was added to precipitate the product. This did not yield anysolid, so the crude product was reconcentrated and purified by PTLC (10%MeOH in DCM) to give, after 2 weeks drying, 0.040 g (46% yield) of SA-84as a yellow-green solid. ¹H NMR (200 MHz, DMSO-d₆) δ 7.50 (s, 1H), 7.44(s, 1H), 7.16 (d, J=2.0 Hz), 7.09 (s, 1H), 7.08 (dd, coupling constantsnot resolvable due to overlapping singlet, 1H), 6.83 (d, J=8.2 Hz, 1H).

Example 26 Synthesis of SA-86

To a mixture of 1-bromo-2-iodo-4,5-dimethoxybenzene (41) (27 mg, 0.078mmol), Pd(PPh₃)₄ (4.1 mg, 5 mol %) and toluene (1 mL) was added thesolution of 2-(3,4-dimethoxyphenyl)-5-(tributylstannyl)thiazole (SA-81)(36 mg, 0.070 mmol) and toluene (1 mL). The reaction was purged withnitrogen and refluxed 16 h. The concentrated reaction mixture waspurified by PTLC (35% EtOAc in hexanes) and afforded 42 (20 mg, 65%yield).

SA-86 was prepared by the reaction of5-(2-bromo-4,5-dimethoxyphenyl)-2-(3,4-dimethoxyphenyl)thiazole (42) (20mg, 0.046 mmol) and BBr₃ (in DDM??) (0.46 mL, 0.46 mmol) according tothe similar procedure for compound SA-58, and obtained SA-86 as a yellowsolid quantitatively.

Example 27 Synthesis of SA-87

2-Bromo-4,5-dimethoxybenzoic acid (3) (4.50 g, 17.6 mmol), veratrole(2.44 g, 17.7 mmol), and 60 g of polyphosphoric acid (PPA) were heatedto 80° C. for 45 min. The reaction mixture turned a deep orange colorduring this time. The crude mixture was treated with 400 mL of water,allowed stir overnight, and the resultant crude product was filtered asa brownish solid. The solid was recrystallized twice from ethanol togive 1.89 g (28% yield) of benzophenone 43. ¹H NMR (CDCl₃, δ) 7.54 (d,J=2.0 Hz, 1H), 7.26 (dd, J=2.0, 8.0 Hz), 7.07 (s, 1H), 6.90-6.83(overlapping peaks, 3H), 3.95 (s, 6H), 3.88 (s, 3H), 3.84 (s, 3H).

Benzophenone 43 (1.31 g, 3.4 mmol) in 10 mL DCM was added to a solutionof 1.00 g NaBH₄ (26 mmol) in 10 mL of TFA. Caution: the reaction betweenNaBH₄ and TFA is extremely exothermic with the evolution of hydrogengas. It is recommended that: (1) an ice bath be used when the additionis occurring and (2) NaBH₄ pellets are used as opposed to powder. Themixture was stirred 16 h, quenched with 15 mL of water, and diluted with20 mL of DCM. The aqueous layer was made basic with 10 N NaOH, thelayers were separated, the aqueous layer extracted with one portion of10 mL DCM, and the organic layer dried with Na₂SO₄ and concentrated.Flash 40+M column chromatography (Biotage) gave 1.15 g (91% yield) of 44as a thick, yellow oil.

¹H NMR (CDCl₃, δ) 7.04 (s, 1H), 6.80 (d, J=8.2 Hz, 1H), 6.74-6.66(overlapping peaks, 2H), 6.63 (s, 1H), 3.99 (s, 2H), 3.86 (s, 6H), 3.84(s, 3H), 3.77 (s, 3H).

Bromide 44 (0.056 g, 0.172 mmol) in 2 mL of DCM was cooled to 0° C. andtreated with 1 mL of 2 M BBr₃ in DCM. The mixture was stirred 2 h whileallowing to warm to ˜23° C., quenched with methanol, and concentrated.The crude, concentrated product was treated with 10 mL of water,extracted twice with eight mL of ethyl acetate, and the organic layerswere concentrated to give the SA-87 as an off-white solid.

¹H NMR (CDCl₃, δ) 6.93 (d, J=8.2 Hz, 1H), 6.62-6.57 (overlapping peaks,2H), 6.51 (bs, 1H), 6.43-6.40 (m, 1H), 3.67 (s, 2H).

Example 28 Synthesis of SA-88 and SA-90

3,4-dimethoxybenzoic acid (1) (3.75 g, 20.6 mmol), veratrole (2.86 g,20.7 mmol), and 80 g of polyphosphoric acid (PPA) were heated to 80° C.with stirring via overhead stirrer for 1 h. Water was added, and themixture was stirred until a solid formed. This solid was filtered,recrystallized twice from ethanol, filtered, and dried to give 3.79 g(61% yield) of benzophenone 64.

¹H NMR (CDCl, δ) 7.43 (d, J=2.0 Hz, 2H), 7.38 (dd, J=2.0, 8.2 Hz, 2H),6.90 (d, J=8.2 Hz, 2H), 3.96 (s, 6H), 3.94 (s, 6H).

Benzophenone 64 (0.50 g, 1.66 mmol) was dissolved in 4 mL of DCM andadded to a solution made from NaBH₄ (0.356 g, 9.4 mmol) and 3 mL TFA.The resultant mixture was stirred 18 h, diluted with 15 mL of DCM and 10mL of water, made basic with 10 N NaOH, and the layers separated. Theaqueous was extracted once more 10 mL of DCM, and the combined organiclayers were dried and concentrated. The crude product was purified byPTLC to give 0.42 g (88% yield) of 65.

The previous reaction was repeated using 1.12 g of benzophenone 64 withthe other reagents scaled appropriately, and gave 0.86 g (81% yield) of65.

¹H NMR (CDCl₃, δ), 6.83-6.78 (m, 2H), 6.75-6.69 (m, 4H), 3.88-3.83(multiple singlets, 14).

A solution of 65 (0.500 g, 1.7 mmol) in 3 mL of AcOH was treated with0.120 g of 69% aq. HNO₃ (1.3 mmol) and stirred for 1.5 h. The mixturewas poured into 50 mL of water, and extracted twice with 25 mL of ethylacetate. The combined organic layers were washed three times with 20 mLof water, dried, and concentrated to give the crude product, which waspurified by PTLC to give 0.123 g of 65 (25% recovery), 0.137 g (24%yield) of 67, and 0.041 g (6% yield) of 66.

¹H NMR of 66 (CDCl₃, δ), 7.65 (s, 2H), 6.54, (s, 2H), 4.66 (s, 2H), 3.93(s, 6H), 3.80 (s, 6H).

¹HMR of 67 (CDCl₃, δ), 7.60 (s, 1H), 6.79-6.63 (overlapping peaks, 4H),4.26 (s, 2H), 3.91-3.81 (m, 12H).

¹³C NMR of 67 (CDCl₃, δ), 153.0, 149.1, 147.7, 147.3, 131.6, 131.3,120.9, 113.6, 112.4, 111.3, 108.2, 56.42, 56.27, 55.9, 38.3.

A mixture of 66 (0.030 g, 0.08 mmol) in 3 mL of DCM was treated with 1mL of 2 M BBr₃ and stirred 2.5 h. Methanol (10 mL) was added to quenchthe reaction, the mixture was concentrated, and water was added. Theresultant brown solid was filtered and not purified further (due to lackof solubility of the product) to give 0.007 g (27% yield) of SA-88. ¹HNMR (DMSO-d₆, δ), 8.87 (bs, 2H), 8.71 (bs, 2H), 7.56 (s, 4H), 3.48 (s,2H).

A mixture of 67 (0.030 g, 0.09 mmol) in 2 mL of DCM was treated with 1mL of 2 M BBr₃ and stirred for 2.5 h. Methanol (10 mL) was added toquench the reaction, the mixture concentrated, and treated with 10 mL ofwater. This was extracted with 10 mL of ethyl acetate, dried, andconcentrated to give 8 mg (32% yield) of SA-90 as a thick brown oil thatslowly solidified.

¹H NMR (DMSO-d₆, δ), 7.54, (s, 3H), 6.97-6.93 (overlapping peaks, 2H),6.50 (d, 1H, overlapping with next peak), 6.45 (d, J=2.0 Hz, 1H), 3.85(s, 2H)

Example 29 Synthesis of SA-93

2-Iodo-4,5-dimethoxybenzoic acid (49) (2.76 g, 9.0 mmol), veratrole(1.20 g, 8.6 mmol), and 50 g of polyphosphoric acid (PPA) were heated to90° C. for 15 min. An additional 0.30 g (2.1 mmol) of veratrole wasadded. The reaction mixture turned a deep orange color during thereaction. The crude mixture was treated with 600 mL of ice water,sonicated, and the resultant crude product was filtered as a brownishsolid (1.55 g, 40% yield). This product 50 was used without furtherpurification.

Benzophenone 50 (1.55 g, 3.6 mmol) in 10 mL DCM was added to a solutionof 2.00 g NaBH₄ (26 mmol) in 10 mL of TFA. Caution: the reaction betweenNaBH₄ and TFA is extremely exothermic with the evolution of hydrogengas. It is recommended that: (1) an ice bath be used when the additionis occurring and (2) NaBH₄ pellets are used as opposed to powder. Themixture was stirred 16 h, quenched with 50 mL of water, and diluted with15 mL of DCM. The aqueous layer was made basic with 10 N NaOH, thelayers were separated, the aqueous layer extracted once with 25 mL DCM,and the organic layer dried with Na₂SO₄ and concentrated. The crudeproduct consisted of the iodo compound 51 and the des-iodo 46. Theproducts practically co-elute when Flash 40+M column chromatography isused, so the crude was purified using PTLC with 0.4% ethyl acetate asthe eluent and eluting the PTLC plates several times to give 0.30 g (20%yield) of 51 as a thick, yellow oil.

¹H NMR (CDCl₃, δ) 6.80 (d, J=8.0 Hz, 1H), 6.74-6.64 (overlapping peaks,4H), 3.98 (s, 2H), 3.85 (bs, 9H), 3.75 (s, 3H).

¹³C NMR (CDCl₃, δ) 149.5, 149.0, 148.1, 147.6, 136.3, 132.6, 121.8,120.8, 113.1, 112.3, 111.3, 88.8, 56.2, 55.9, 45.6.

Iodide 51 (0.150 g, 0.36 mmol) in 40 mL of DCM was cooled to −78° C. andtreated with BBr₃ (0.75 g, 3 mmol) neat. The mixture was stirred 2 h,allowed to warm to ˜23° C., stirred 3 h more, quenched with water,extracted with 20 mL ethyl acetate, and the organic dried andconcentrated. The crude, concentrated product was precipitated with DCM,and filtered to give 0.022 g of SA-93 (17% yield).

¹H NMR (DMSO-d₆, δ) 6.62-6.52 (overlapping peaks, 3H), 6.50 (d, J=1.9Hz, 1H), 6.41 (dd, J=2.0, 8.2 Hz, 1H), 3.54 (s, 2H), 4.5-3.5 (broadpeak, 4H, “—OH”)

Example 30 Synthesis of SA-94 and SA-98

A mixture of 3′,4′-dimethoxyacetophenone (52) (0.54 g, 3 mmol) and3,4-dimethoxybenzaldehyde (0.51 g, 3.06 mmol) (30) in 15 mL of absoluteethanol was treated with 1.75 g of NaOH, sonicated for 10 min, thenstirred overnight. The resultant mixture was cooled to 0° C., filtered,and washed with 4° C. ethanol to obtain a yellow solid that was dried togive 0.88 g of 53 (89% yield).

¹H NMR (CDCl₃, δ) 7.76 (d, J=15.4 Hz, 1H), 7.68 (dd, J=2.0, 8.4 Hz, 1H),7.62 (d, J=2.0 Hz, 1H), 7.40 (d, J=15.4 Hz, 1H), 7.19 (dd, J=2.0, 8.4Hz, 1H), 6.93 (d, J=8.0 Hz, 1H), 6.90 (d, J=8.0 Hz, 1H).

A solution of 53 (0.14 g, 0.43 mmol) was dissolved in 40 mL of DCM,cooled to −78° C., and BBr₃ (0.98 g, 3.9 mmol) in 4 mL of DCM was added.The mixture was allowed to warm to 0° C. over 2 h, and then immediatelywarmed to 23° C. The mixture was stirred 2 h, quenched with 20 mL ofwater, and extracted with 100 mL of ethyl acetate. The organic layer wasdried, concentrated until ˜5 mL remained, and treated with 30 mL of DCM.A deep purple solid precipitated from the solution. This solid wasfiltered, and dried to a yellow-green solid in the dark to give 0.068 g(59% yield) of SA-94.

¹H NMR (DMSO-d₆) 10.5-8.0 (bs, 4H), 7.57-7.47 (m, 2H), 7.49 (s, 2H),7.20 (bs, 1H), 7.12 (m, 1H), 6.84 (d, J=8.4 Hz, 1H), 6.79 (d, J=8.6 Hz,1H).

A solution of 53 (0.082 g, 0.25 mmol) in 1.5 mL of acetic acid wastreated with 0.5 mL of hydrazine hydrate and heated to 135° C. for 2 hand 140° C. for 1 h. The yellow color of the starting material graduallydischarged throughout the course of the reaction. The crude reactionmixture was poured into 20 mL of water, extracted with 10 mL of ethylacetate twice. The combined organic layers were washed twice with 20 mLof water, dried, and concentrated to give 0.058 g (61% yield) of 54 as aclear oil that was not purified further.

A solution of 54 (0.056 g, 0.59 mmol) in 20 mL of DCM was cooled to −78°C., and treated with BBr₃ (1.025 g, 4.1 mmol) in 5 mL of DCM. Themixture was stirred for 3 h at −78° C., stirred for 1 h at ˜0° C., andquenched with water. The resultant mixture was extracted with 100 mL ofethyl acetate, and the organic layer was dried and concentrated to givethe crude product which was taken up in 2 mL of ethyl acetate, treatedwith 2 mL of hexanes, and treated with 50 mL of DCM. The precipitatedproduct SA-98 (0.019 g, 40% yield) was collected by filtration and driedto give an off-white powder.

¹H NMR (DMSO-d₆) 9.4 (bs, 1H), 9.2 (bs, 1H), 8.9 (bs, 1H), 8.8 (bs, 1H),7.26 (d, J=2.2 Hz, 1H), 6.98 (dd, J=2.2, 8.4 Hz, 1H), 6.77 (d, J=8.2 Hz,1H), 6.64 (d, J=8.4 Hz, 1H), 6.52 (d, J=2.2 Hz, 1H), 6.44 (dd, J=2.2,8.0 Hz, 1H), 5.32 (dd, J=3.6, 11.0 Hz, 1H), 3.4 (m, 1H), 3.22 (m, 1H),2.24 (s, 3H).

HRMS, Calculated for C₁₇H₁₇N₂O₅ (M+H)⁺ 329.1137. found 329.1125.

Example 31 Synthesis of SA-95

A mixture of 3′,4′-dimethoxyacetophenone (52) (2 mmol, 0.36 g) and6-bromoveratraldehyde (55) (2 mmol, 0.49 g) in 15 mL of ethanol wastreated with 1 g of NaOH and 0.015 g (2.78 mmol) of NaOMe. The mixturewas stirred at 23° C. for 18 h, cooled to 4° C., and the resultantyellow solid filtered to give the bromochalcone 56 as a yellow solid(0.68 g, 83% yield).

¹H NMR (CDCl₃, δ), 8.07 (d, J=15.6 Hz, 1H), 7.75 (m, 1H), 7.67 (d, J=1.8Hz, 1H), 7.38 (m, 1H), 7.22 (s, 1H), 7.12 (s, 1H), 6.95 (d, J=8.2 Hz,1H), 3.98-3.94 (overlapping singlets, 12H).

A mixture of bromochalcone 56 (0.20 g, 0.49 mmol) in 30 mL of DCM wascooled to −78° C. and treated with BBr₃ (0.72 g, 2.88 mmol) in 4 mL ofDCM. The mixture was stirred at −78° C. for 2.5 h, warmed gradually to0° C. over 1 h, warmed to 23° C., and stirred an additional 1 h. Thereaction was quenched with 20 mL of water, and the mixture extractedwith 50 mL of ethyl acetate. The ethyl acetate layer was washed oncewith 20 mL of brine, dried concentrated to 2 mL, and treated with 50 mLof DCM. The product was filtered and dried to get SA-95 as a reddishsolid (0.056 g, 33% yield).

¹H NMR (dmso-d₆) 10.2 (bs, 1H), 9.8 (bs, 1H), 9.3 (bs, 2H), 7.81 (d,J=15.4 Hz, 1H), 7.61 (d, J=2.2 Hz, 1H), 7.56-7.49 (overlapping peaks,3H), 7.05 (s, 1H), 6.86 (d, J=8.2 Hz, 1H).

Example 32 Synthesis of SA-96

A mixture of 2′-bromo-4′,5′-dimethoxyacetophenone (57) (0.390 g, 1.5mmol) and 3,4 dimethoxybenzaldehyde (30) (0.310 g, 1.9 mmol) in 10 mL ofethanol was treated with 0.75 g of NaOH. The mixture was inverted tentimes to thoroughly mix the starting materials, sonicated 5 min, allowedto stand 2 h, and sonicated 5 min again. A yellow precipitate resulted,and this was filtered and dried to give bromochalcone 58 as a lightyellow solid (0.482 g, 79% yield).

¹H NMR (CDCl₃, δ) 7.41 (d, J=2.2 Hz, 1H), 7.39-7.34 (m, 3H), 7.25 (s,1H), 7.17 (d, J=16.0 Hz, 1H), 7.10 (s, 1H), 7.00 (d, J=8.4 Hz, 1H),3.86-3.81 (overlapping singlets, 12H).

¹³C NMR (CDCl₃, δ) 194.0, 151.9, 151.1, 149.5, 148.5, 146.6, 133.4,127.6, 124.6, 124.0, 116.5, 112.8, 112.1, 111.5, 110.2, 56.6, 56.4,56.2, 56.1.

A mixture of bromochalcone 58 (0.27 g, 0.66 mmol) in 30 mL of DCM wascooled to −78° C., and treated with 1.25 g (5 mmol) of BBr₃ in 5 mL ofDCM. The mixture was stirred at −78° C. for 3 h, warmed immediately to23° C., stirred at 23° C. for 1 h, quenched with 20 mL of water, andextracted with 125 mL of ethyl acetate. The organic layer was dried andconcentrated to 1 mL. The concentrate was treated with an excess of DCMto precipitate a purplish-red solid that was filtered and dried to giveSA-96 as a reddish-brown solid (0.036 g, 16% yield).

¹H NMR (DMSO-d₆, δ) 7.26 (d, J=16.0 Hz, 1H), 7.09 (bs, 1H), 7.05-6.86(overlapping peaks, 4H), 6.77 (d, J=8.2 Hz, 1H).

Example 33 Synthesis of SA-97

A mixture of 2,4,5-trimethoxybenzoic acid (59) (1.06 g, 5 mmol),veratrole (0.69 g, 5 mmol), and 10 g of PPA was heated with a heat gunand stirred with a stirring rod for 20 min. Water (100 mL) was added tothe reaction mixture and the resultant mixture was cooled to 4° C. andstirred with a stirring rod for 20 min. The water was decanted and theresultant grey, gummy solid was taken up in a hot solution 20 mL ofreagent alcohol and 10 mL of water. The mixture was cooled and filteredto give 0.43 g of orange crystals. Water (40 mL) was added to thefiltrate and the resultant orange solid was filtered to give 0.72 g ofamorphous orange solid. Both fractions were the desired 60 (1.15 g, 69%yield).

A mixture of benzophenone 60 (0.93 g, 2.9 mmol) in 20 mL of DCM wasadded to 0.92 g of NaBH₄ (24.3 mmol) in 10 mL of TFA. Caution: thereaction between NaBH₄ and TFA is extremely exothermic with theevolution of hydrogen gas. It is recommended that: (1) an ice bath beused when the addition is occurring and (2) NaBH₄ pellets are used asopposed to powder. The mixture was stirred for 6 h, and an additionalportion of NaBH₄ (0.53 g, 14.0 mmol) was added. The mixture was stirred20 h, diluted with 50 mL of DCM and 50 mL of water, and the aqueouslayer made basic with NaOH. The layers were separated, and the aqueouslayer was extracted once with 50 mL of DCM. The organic layers werecombined, dried, and concentrated to give a yellow oil that was purifiedby Flash 40+M chromatography (Biotage) to give 61 as a pale yellow,thick oil (0.63 g, 71% yield).

¹H NMR (CDCl₃, δ) 6.83-6.76 (overlapping peaks, 3H), 6.66 (s, 1H), 6.57(s, 1H), 3.91-3.86 (overlapping singlets, 11H), 3.83 (s, 3H), 3.79 (s,3H).

A mixture of pentamethoxy compound 61 (0.262 g, 0.82 mmol) in 30 mL ofDCM at −78° C. was treated with BBr₃ (2.00 g, 8 mmol) in 8 mL of DCM.The resultant mixture was stirred at 78° C. 1 h, warmed to 23° C.immediately, stirred at 23° C. for 4 h, quenched with 20 mL of water andextracted with 100 mL of ethyl acetate. The ethyl acetate layer wasconcentrated to dryness, taken up in approximately 0.5 mL of ethylacetate, precipitated with DCM, cooled to −20° C., and filtered to giveSA-97 as a white solid (0.93 g, 46% yield).

¹H NMR (DMSO-d₆) 8.63-8.29 (bs, 4H), 6.58 (d, J=7.8 Hz, 1H), 6.52 (d,J=2.0 Hz, 1H), 6.40 (dd, J=2.0, 8.0 Hz, 1H), 6.29 (s, 1H), 6.26 (s, 1H),3.50 (s, 2H).

Example 34 Synthesis of SA-99

A mixture of dibromide 62 (0.562 g, 2 mmol), boronic acid 15 (0.910 g, 5mmol), 20 mL of dioxane, and 24 mL of 2 M Na₂CO₃ (aqueous) was degassedby nitrogen purge. Then Pd(PPh₃)₄ (0.12 g, 0.10 mmol) was added. Theresultant mixture was refluxed 20 h, diluted with 50 mL of water, andextracted three times with 50 mL of ethyl acetate. The combined organiclayers were washed once with 50 mL of water, dried, and concentrated togive the crude product. This was purified by Flash 40+M chromatography(Biotage) using gradient ethyl acetate in hexanes as the eluent to give63 as a yellow solid (0.288 g, 36% yield).

¹H NMR (CDCl₃, δ), 7.94 (d, J=1.8 Hz, 1H), 7.75 (dd, J=1.8, 8.0 Hz, 1H),7.47 (d, J=8.0 Hz, 1H), 7.17 (dd, J=2.2, 8.2 Hz, 1H), 7.10 (d, J=2.0 Hz,1H), 6.96 (d, J=8.0 Hz, 1H), 6.90-6.85 (overlapping peaks, 3H), 3.95 (s,3H), 3.92 (s, 3H), 3.90 (s, 3H), 3.87 (s, 3H).

¹³C NMR (CDCl₃, δ) 149.9, 149.7, 149.6, 149.3, 149.1, 141.1, 133.9,132.2, 131.2, 130.0, 129.5, 121.8, 120.5, 119.6, 111.7, 111.4, 111.2,110.1, 56.11, 56.05, 56.0, 55.9.

A solution of 63 (0.062 g, 0.16 mmol) in 25 mL DCM was cooled to −78°C., treated with BBr₃ (1.125 g, 4.5 mmol) in 6 mL of DCM, stirred 2.5 hat −78° C., warmed to 23° C., stirred at −23° C. for 1.5 h, quenchedwith 20 mL water, and extracted once with 100 mL of ethyl acetate. Theethyl acetate layer was dried and concentrated to 3 mL and precipitatedwith excess DCM. The precipitate was filtered and dried to give SA-99 asa yellow solid (0.037 g, 70% yield).

¹H NMR (DMSO-d₆) 9.17-9.11 (bs, 4H), 7.96 (d, J=1.6 Hz, 1H), 7.83 (dd,J=2.2, 8.2 Hz, 1H), 7.49 (d, J=8.0 Hz, 1H), 7.14 (bs, 1H), 7.08 (d,J=8.4 Hz, 1H), 6.88-6.79 (overlapping peaks, 2H), 6.72 (d, J=2.2 Hz,1H), 6.63 (dd, J=2.0, 8.2 Hz, 1H),

HRMS Calculated for C₁₈H₁₃NO₆Na (M+Na) 362.0641. Found 362.0645.

Example 35 Synthesis of SA-108

To 1.00 g (4.07 mmol) of acid chloride 68 in 25 mL of DCM at 0° C. wasadded 5 mL of pyridine. The mixture was stirred 3 min, and 0.76 g (3.87mmol) of known hydrazide 69 was added at once. The mixture was graduallyallowed to warm to 21-23° C. and stirred at this temperature for 16 h.The mixture was concentrated, treated with 10 mL of ethanol, warmed toreflux, and diluted with 30 mL of water. After returning the mixture toreflux, ethanol was added gradually until a solution formed. Thesolution was allowed to cool, and the precipitated light yellow solidwas collected and dried to give 0.498 g (1.24 mmol, 32% yield) of 70.

¹H NMR (CDCl₃ and MeOD, δ) 7.54 (s, 1H), 7.45 (dd, J=2.2, 8.4 Hz, 1H),7.39 (d, J=1.8 Hz), 7.16 (s, 1H), 6.82 (d, J=8.4 Hz, 1H), 3.98-3.73(overlapping singlets+residual H₂O peak, 12H).

A mixture of 0.398 g (1 mmol) of 70 and 0.452 g (1.11 mmol) ofLawesson's reagent in 50 mL of THF were refluxed for 16 h. The mixturewas concentrated and purified by PTLC using 10% EtOAc in DCM as theeluent to give 0.255 g (0.62 mmol, 62% yield) 71 as a bright yellowsolid.

¹H NMR (DMSO-d₆) 7.77 (s, 1H), 7.60-7.58 (overlapping peaks, 2H), 7.40(s, 1H), 7.15 (d, J=8.8 Hz, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.89 (s,3H), 3.87 (s, 3H).

A mixture of 0.11×) g (0.24 mmol) of 71 in 5 g of pyridine hydrochloridewas heated to 200° C. for 30 min. The mixture was cooled, treated with30 mL of water, and filtered. The resultant solid was recrystallizedfrom aqueous methanol to yield 0.016 g of a brown solid as SA-108. Theproduct is only sparingly soluble in DMSO-d₆, and insoluble in otherdeuterated solvents or solvent mixtures.

¹H NMR (DMSO-d₆, δ), 8.79 (s, 1H), 7.80 (bs, 1H), 7.57 (s, 1H), 7.43 (m,1H), 7.28 (m, 1H), 6.91 (s, 1H), 6.80 (d, J=8.0 Hz, 1H). Example1—Compounds provided herein bind are potent disruptors/inhibitors ofParkinson's disease α-synuclein fibrils

Example 36 Compounds Provided Herein are Potent Disruptors/Inhibitors ofParkinson's Disease α-Synuclein Fibrils

The compounds were found to be potent disrupters/disaggregators ofα-synuclein fibrils. In this set of studies, the efficacy of certaincompounds provided herein to cause adisassembly/disruption/disaggregation of pre-formed fibrils ofParkinson's disease (i.e. consisting of α-synuclein fibrils) wasanalyzed. For the studies described below in Parts A and B, 69 μM ofα-synuclein (rPeptide, Bogart, CA) was first incubated at 37° C. for 4days in 20 mM sodium acetate buffer at pH 4 with circular shaking (1,300rpm) to cause α-synuclein aggregation and fibril formation.

Part A: Thioflavin T Fluorometry Data

In one study, Thioflavin T fluorometry was used to determine the effectsof the compounds on α-synuclein fibrils. In addition to test compounds,this experiment included a positive control compound and a negativecontrol compound for reference. In this assay Thioflavin T bindsspecifically to fibrillar amyloid, and this binding produces afluorescence enhancement at 485 nm that is directly proportional to theamount of fibrils formed. The higher the fluorescence, the greater theamount of fibrils formed (Naki et al., Lab. Invest. 65:104-110, 1991;Levine Protein Sci. 2:404-410, 1993; Amyloid: Int. J. Exp. Clin. Invest.2:1-6, 1995).

Following initial α-synuclein fibrilization as described above, 6.9 μMα-synuclein was incubated at 37 C for 2 days with shaking (1,300 rpm),either alone, or in the presence of one of the compounds (at testcompound:α-synuclein molar ratios of 50:1, 10:1, 5:1, 1:1, 0.5:1, 0.1:1,0.05:1 and 0.01:1) in phosphate-buffered saline, pH 7.4+0.02% sodiumazide. Following 2 days of co-incubation, 50 μl of each incubationmixture was transferred into a 96-well microliter plate containing 150μl of distilled water and 50 μl of a Thioflavin T solution (i.e. 500 μMThioflavin T in 250 μM phosphate buffer, pH 6.8). The finalconcentration of Thioflavin T reagent is 100 μM in 50 μM phosphatebuffer, pH 6.8. The fluorescence was read at 485 nm (444 nm excitationwavelength) using an ELISA plate fluorometer after subtraction withbuffer alone or compound alone, as blank.

The results of the 2-day incubations are presented below. For eachcompound, the % inhibition of Thioflavin T fluorescence (i.e. thedecrease compared to control reactions containing α-synuclein alone) wasplotted against the log of the concentration of the test compound(expressed as mole ratio relative to α-synuclein). Where possible, theeffective concentration of SA compound that yields 50% of maximal %decrease of Thioflavin T fluorescence (EC₅₀) was calculated from thesigmoidal shaped dose response curve. The compounds (SA-52, SA-53,SA-54, SA-55, SA-57, SA-58, SA-59, SA-61, SA-62, SA-63, SA-64, SA-66,SA-67, SA-68, SA-69, SA-70, SA-72, SA-73, SA-93, SA-94, SA-95, SA-96,SA-97, SA-98 and SA-99 and positive reference compound #1) all caused adose-dependent and extensive disruption/disassembly of preformedα-synuclein fibrils (Table 1). For example, compound SA-57 caused asignificant (p<0.01 relative to α-synuclein alone) nearly completeinhibition (97-100%) when used at test compound:α-synuclein molar ratios≧5:1 and a significant 83% inhibition when used at a testcompound:α-synuclein molar ratio of 1:1 (FIG. 1), whereas the negativereference compound showed no significant inhibition of Thioflavin Tfluorescence at any of the concentrations tested (not shown). The EC₅₀of SA-57 for inhibition of Thioflavin T fluorescence was determined tobe 0.23 moles of test compound per mole of α-synuclein (Table 1). Forthe compounds described here that caused a dose-dependentdisruption/disassembly of α-synuclein fibrils, the maximum % inhibitionranged from 76-100% and the EC₅₀ values ranged from 0.08-4.3 moles oftest compound:α-synuclein, with most compounds showing highly potenteffects (i.e. EC₅₀<1 mole of test compound:α-synuclein) in this assay.

This study indicated that the compounds provided herein are potentdisrupters/dissaggregators of Parkinson's disease α-synuclein fibrils,and usually exert their effects in a dose-dependent manner.

TABLE 1 SA compounds disrupt/disaggregate α-synuclein aggregates asmeasured by Thioflavin T fluorometry. SA # EC₅₀ (mole ratio) Max %Decrease 52 0.28 99 53 0.17 100 54 0.74 98 55 0.08 92 57 0.23 100 580.19 100 59 0.58 96 61 0.10 100 62 1.09 95 63 0.86 100 64 0.39 100 660.69 100 67 4.3 76 68 0.77 100 69 0.73 88 70 0.17 100 72 0.12 100 730.52 100 93 0.16 100 94 0.15 100 95 0.16 100 96 0.14 100 97 0.16 99 980.65 100 99 0.65 100 Positive Reference #1 0.24 100 Negative Referencenot determined 0

Part B: Congo Red Binding Data

In the Congo red binding assay, the ability of a given test compound toalter α-synuclein aggregate binding to Congo red is quantified. In thisassay Congo red binds specifically to fibrillar amyloid, and thisbinding is directly proportional to the amount of fibrils formed.Following initial α-synuclein fibrilization as described above,α-synuclein aggregates and test compounds were incubated for 2 days andthen vacuum filtered through a 0.2 μm filter. The amount of α-synucleinretained in the filter was then quantitated following staining of thefilter with Congo red. After appropriate washing of the filter, anylowering of the Congo red color on the filter in the presence of thetest compound (compared to the Congo red staining of the amyloid proteinin the absence of the test compound—i.e. α-synuclein alone) wasindicative of the test compound's ability to diminish/alter the amountof aggregated and congophilic α-synuclein and thus causedisassembly/disruption/disaggregation of α-synuclein fibrils.

In one study, the ability of α-synuclein fibrils to bind Congo red inthe absence or presence of increasing amounts of the compounds providedherein, including a positive and a negative reference compound (at testcompound:α-synuclein molar ratios of 50:1, 10:1, 5:1, 1:1, 0.5:1, 0.1:1,0.05:1, 0.01:1) was determined. The results of 2-day incubations arepresented in Table 2 below. Whereas the negative reference compoundcaused no significant inhibition of α-synuclein fibril binding to Congored at all concentrations tested (not shown), the test compounds causeda dose-dependent inhibition of α-synuclein binding to Congo red. Forexample, compound SA-57 caused a significant (p<0.01) inhibition (66%)when used at a test compound:α-synuclein molar ratio of 50:1 and asignificant 49% inhibition when used at a test compound:α-synucleinmolar ratio of 5:1 (FIG. 2). The EC₅₀ of SA-57 for inhibition of CongoRed binding was determined to be 2.0 moles of test compound per mole ofα-synuclein (Table 2). For the compounds described here that caused adose-dependent disruption/disassembly of α-synuclein fibrils, themaximum % inhibition ranged from 25-94% and the EC₅₀ values ranged from1.0-15.9 moles of test compound per mole of α-synuclein. Taken together,the results of this study indicated that compounds of this inventiondisrupt/disaggregate/disassemble α-synuclein aggregates as indicated bytheir ability to inhibit Parkinson's disease type α-synuclein fibrilbinding to Congo red, and usually exert their effects in adose-dependent manner.

TABLE 2 SA compounds disrupt/disaggregate α-synuclein fibrils/aggregatesas measured by Congo Red binding assay. SA # EC₅₀ (mole ratio) Max %Decrease 52 10.7 68 53 6.8 75 54 15.9 57 55 4.5 38 57 2.0 66 58 1.9 9459 7.6 85 61 2 84 63 9.5 72 64 2.3 85 66 2.2 48 67 6.8 25 68 6.4 60 6914.2 39 70 3.4 88 72 2.9 29 73 1.1 35 93 1.9 62 94 4.5 65 95 4.5 64 965.1 72 97 1.0 40 98 10.1 44 99 14.2 54 Positive Reference #1 4.5 66Negative Reference not determined 0

Example 37 Compounds of this Invention are Potent Disruptors/Inhibitorsof α-Synuclein Fibrils and/or Aggregates Associated with Parkinson'sDisease

Parkinson's Disease is characterized by the accumulation of insolubleintraneuronal aggregates called Lewy Bodies, a major component of whichis α-synuclein (reviewed in Dauer et al., Neuron, 39:889-909, 2003).Since autosomal dominant mutations in α-synuclein cause a subset offamilial Parkinson's disease, and since these mutations increase thelikelihood of α-synuclein to aggregate and form Lewy Bodies, aggregatedα-synuclein is proposed to be directly involved in the etiology anddisease progression (Polymeropoulos et al., Science 276:1197-1199, 1997;Papadimitriou et al., Neurology 52:651-654, 1999). Structural studieshave revealed that intracellular Lewy bodies contain a large proportionof misfolded proteins with a high degree of β-pleated sheet secondarystructure. Therefore, since many of the compounds described herein causedisassembly/disruption/disaggregation of α-synuclein aggregates in thein vitro assays (Thioflavin T fluorometry and Congo Red binding assays)described above, studies were also conducted in living cells todetermine the efficacy of these compounds to inhibit or preventα-synuclein aggregation associated with Parkinson's disease.

To test the therapeutic potential of the compounds, two cell-basedassays were utilized. In both assays, rotenone is used to inducemitochondrial oxidative stress and cause α-synuclein aggregation. Thefirst assay utilizes the binding of the fluorescent dye Thioflavin S tostructures with high β sheet content, including α-synuclein fibrils.Therefore, quantitative assessment of the extent of ThioflavinS-positive staining of fixed cells is used to test the ability of thetest compounds to inhibit/prevent or decrease the amount of α-synucleinaggregates relative to cells that were treated with rotenone only. Inthe second assay, cell viability is assessed using the XTT cytotoxicityassay (Cell Proliferation Assay Kit II, Roche, Mannheim, Germany), whichis dependent on intact, functional mitochondria in live cells. Thus, theXTT cytotoxicity assay is used to test the ability of the compounds toameliorate the mitochondrial toxicity and resulting loss of viabilityassociated with the accumulation of α-synuclein aggregates. Thesestudies are presented in the following examples.

To carry out these studies, a cell culture model was used in which humanα-synuclein aggregation is experimentally induced. BE-M17 humanneuroblastoma cells stably transfected with A53T-mutant humanα-synuclein were obtained. Cell culture reagents were obtained fromGibco/Invitrogen, and cells were grown in OPTIMEM supplemented with 10%FBS, Penicillin (100 units/ml), Streptomycin (100 μg/ml) and 500 μg/mlG418 as previously described (Ostrerova-Golts et al., J. Neurosci.,20:6048-6054, 2000).

Thioflavin S is commonly used to detect aggregated protein structures insitu, including in brain tissue (Vallet et al., Acta Neuropathol.,83:170-178, 1992), and cultured cells (Ostrerova-Golts et al., J.Neurosci., 20:6048-6054, 2000), whereas Thioflavin T is often used as anin vitro reagent to analyze the aggregation of soluble proteins intofibrils enriched in β-pleated sheet structures (LeVine III, Prot. Sci.,2:404-410, 1993). Therefore, Thioflavin S histochemistry was used oncultured cells to detect aggregates containing a high degree ofβ-pleated structures that formed in response to oxidativestress-inducing agents (in this case rotenone) as previously described,with minor modifications (Ostrerova-Golts et al., J. Neurosci.,20:6048-6054, 2000). Briefly, for these studies cells were grown onPoly-D-Lysine coated glass slide chambers at approximately 4.5-5.5×10⁴cells/cm². After 16-18 hours, cells were treated with 500 nM or 2 μMrotenone (Sigma) or vehicle (0.05% DMSO) as indicated. Within 15 minutesof rotenone (or vehicle) addition, compounds were added at the indicatedconcentration, or mock-treatment was performed in which cell culturemedia only (no compound) was added. Identical treatments were repeatedafter 48 hours. After an additional 24 hours, cells were fixed for 25minutes in 3% paraformaldehyde. After a PBS wash and a deionized waterwash, the cells were incubated with 0.015% Thioflavin S in 50% ethanolfor 25 minutes, washed twice for four minutes in 50% ethanol and twicefor five minutes in deionized water and then mounted using anaqueous-based mountant designed to protect against photobleaching.Aggregates that bind to Thioflavin S were detected with a fluorescentmicroscope using a High Q FITC filter set (480 to 535 nm bandwidth) anda 20× objective lens unless otherwise indicated. Between 8 and 20(usually 16-18) representative images per condition were selected andimaged using Q Capture software by an experimenter who was blinded totreatment conditions. To assess the amount of Thioflavin 5-positiveaggregates, the total area per field covered by Thioflavin S-positiveinclusions was determined by image analysis and quantitation. For thispurpose, background fluorescence that failed to exceed pre-set size orpixel intensity threshold parameters was eliminated using Image Pro Plussoftware. Spurious, non-cell associated fluorescence was manuallyremoved. Unless indicated otherwise, comparisons between groups weremade by comparing mean relative amounts of Thioflavin S-positiveinclusions for a given treatment condition (i.e. cells treated withrotenone only versus cells treated with rotenone and test compound at agiven concentration). Statistical analyses were performed with GraphPadPrism (GraphPad Inc). Differences between means (two samples) wereassessed by the Student's t test. Differences among multiple means wereassessed by one-factor ANOVA followed by Dunnett's post hoc test,compared to rotenone only treated cells. The data presented in Table 3represent statistically significant (p<0.05) reductions (reported aspercent inhibition) in Thioflavin S fluorescence in cells treated withtest compound and rotenone relative to cells treated with rotenone only.

To validate the ability of the assay to quantitatively detect aggregatesthat bind Thioflavin S, staining of BE-M17 cells overexpressing A53Tα-synuclein was carried out and the results revealed a rotenonedose-dependent increase in Thioflavin S-positive aggregates relative tovehicle-treated control cells (FIG. 3). Higher magnification imagesobtained with a 40× objective indicated that the Thioflavin S-positiveaggregates were intracellular and cytoplasmic, analogous to theaccumulation of intracytoplasmic Lewy bodies that are pathologicalhallmarks associated with Parkinson's disease (not shown). Quantitationof the area covered by Thioflavin-S-positive aggregates established that500 nM and 2 μM rotenone were sufficient to induce robust aggregation(FIG. 3) and thus are effective doses to test the ability of compoundsto attenuate the formation of these aggregates.

Using the protocol described above, several selected compounds weretested for their ability to reduce, inhibit, prevent or eliminateThioflavin S-positive aggregates in rotenone-treated BE-M 17 cellsoverexpressing A53T α-synuclein. Examples of results obtained fromexperiments using these compounds are summarized in Table 4. Many of thecompounds tested significantly disrupted, prevented or inhibitedα-synuclein aggregation and fibril formation in the presence of rotenoneas indicated by a decrease in Thioflavin S-positive inclusions, relativeto cells treated with rotenone only. For example, cells treated with 500nM rotenone only exhibited a robust presence of Thioflavin S-positiveaggregates (not shown), whereas addition of 500 nM, 1 μM or 5 μM SA-72markedly reduced the abundance of these rotenone-induced aggregates by63%, 65% and 83%, respectively, relative to rotenone only-treated cells(Table 3). Similarly, in cells treated with 2 μM rotenone only, therewas a robust presence of Thioflavin S-positive aggregates (not shown),whereas addition of 500 nM or 1 μM SA-72 markedly reduced the abundanceof these rotenone-induced aggregates by 67% and 42%, respectively,relative to rotenone only-treated cells (Table 3). Therefore, SA-72reduced, inhibited, prevented and/or eliminated Thioflavin S-positiveaggregates in cells that express human A53T α-synuclein.

In addition to SA-72, compounds SA-52, SA-53, SA-54, SA-58, SA-59,SA-61, SA-62, SA-66, SA-67, SA-68, SA-93, SA-94, SA-95, SA-96 and SA-98,at given concentrations, all showed significantdisruption/prevention/inhibition of rotenone-induced ThioflavinS-positive inclusions when tested in a similar fashion. These resultsare summarized in Table 3.

Taken together, we concluded that the tested compounds SA-52, SA-53,SA-54, SA-58, SA-59, SA-61, SA-62, SA-66, SA-67, SA-68, SA-72 SA-93,SA-94, SA-95, SA-96 and SA-98 effectively and potently reduced,prevented and/or inhibited the formation, deposition and/or accumulationof α-synuclein aggregates in A53T α-synuclein-expressing BE-M17 cells.

TABLE 3 SA compounds prevent/inhibit rotenone-induced ThioflavinS-positive α-synuclein aggregates in cells. Concentration in μM EfficacySA # rotenone compound % Inhibition 52 2 5 49 53 2 2 71 54 0.5 0.5 800.5 2 83 2 1 56 58 0.5 2 68 2 2 63 59 0.5 1 69 0.5 2 76 0.5 5 56 2 1 6761 0.5 0.5 36 0.5 5 48 62 0.5 0.5 81 0.5 1 55 0.5 5 85 66 0.5 0.5 39 0.51 32 0.5 5 94 2 0.5 57 2 1 69 67 0.5 0.5 75 0.5 1 48 0.5 2 92 68 0.5 169 0.5 5 73 2 5 44 72 0.5 0.5 63 0.5 1 65 0.5 5 83 2 0.5 67 2 1 42 93 22 45 94 0.5 2 85 2 0.5 70 2 1 63 95 0.5 0.5 73 0.5 1 98 0.5 2 90 2 0.595 96 0.5 1 82 2 0.5 73 2 2 81 98 0.5 0.5 74 2 1 76 Positive Ref. #1 0.50.5 47 0.5 1 65 0.5 2 70 2 0.5 49 2 1 56 2 2 60 Negative Ref. 0.5 5 nonedetected 2 5 none detected

Example 38 Compounds of this Invention are Neuroprotective AgainstRotenone-Induced Cytotoxicity

The XTT cytotoxicity assay (Cell Proliferation Assay Kit II) waspreviously used to demonstrate that A53T α-synuclein potentiates celldeath in BE-M17 cells through an oxidative stress-dependent mechanism(Ostrerova-Golts et al., J. Neurosci., 20:6048-6054, 2000). Research hasshown that the accumulation of α-synuclein fibrils in Lewy bodiescontributes mechanistically to the degradation of neurons in Parkinson'sdisease and related disorders (Polymeropoulos et al., Science276:2045-2047, 1997; Kruger et al., Nature Genet. 18:106-108, 1998).Here, the XTT Cell Proliferation Assay Kit II (hereafter referred to asthe XTT assay) was used to measure the ability of compounds to provideneuroprotection against rotenone-induced cytotoxicity. The assay isbased on the principle that conversion of the yellow tetrazolium saltXTT to form an orange formazan dye (that absorbs light around 490 nm)occurs only in metabolically active, viable cells. Therefore, lightabsorbance at 490 nm is proportional to cell viability. For this assay,cells were plated in 96 well tissue culture dishes at 10⁴ cells perwell. After 16-18 hours, cells were treated with 500 nM rotenone, orvehicle (0.05% DMSO) as indicated. Approximately 15 minutes afterrotenone addition, compounds were added at the indicated concentration.As a control, compounds were added without rotenone (in the presence of0.05% DMSO vehicle) and resulted in no toxicity at the doses presented.Mock-treatment consisted of cell culture media only (no compound), inthe presence or absence of rotenone. After 44-46 hours of treatment,conditioned media was removed and replaced with 100 μl fresh media and50 μl XTT labeling reaction mixture according to the manufacturer'srecommendations. Five to six hours later, the absorbance at 493 nm wasmeasured and corrected for absorbance at the 620 nm referencewavelength. Treatment with 500 nM rotenone decreased viability by30-40%. Percent inhibition of cell death was calculated as theproportion of the rotenone-induced absorbance (viability) decrease thatwas eliminated by SA compound treatment.

Using the protocol described above, several selected compounds weretested for their ability to provide neuroprotection against therotenone-induced loss of cell viability (cell death) in A53Tα-synuclein-expressing BE-M17 cells. In this series of experiments,there was a 30-40% loss of viability (cell death) in 500 nMrotenone-treated cells, relative to vehicle-treated cells, as expected.However, treatment with the compounds SA-52, SA-58, SA-59, SA-60, SA-61,SA-62, SA-64, SA-67, SA-68, SA-69, SA-70, SA-72, SA-73, SA-93, SA-94,SA-95, SA-96, SA-97, SA-98, and both positive reference compounds 2 and3, resulted in a significant, dose-dependent inhibition ofrotenone-induced cell death. To define the relative potency of eachcompound, the % inhibition of cell death was plotted against the log ofthe dose (μM), and, when possible, the 50% inhibitory concentration(IC₅₀) was calculated from the dose response curve. For example,treatment with 3.5 μM SA-94 resulted in 35% inhibition of cell death andtreatment with 15 μM SA-94 resulted in 58% inhibition of cell death,with a calculated IC₅₀ of 2.3 μM (FIG. 4), whereas treatment with thenegative reference compound showed no inhibition of rotenone-inducedcell death in this dose range (not shown). The positive results fromthis assay for the compounds described herein are summarized in Table 4.

Taken together, we concluded that the tested compounds that wereefficacious in inhibiting rotenone-induced cytotoxicity demonstrateneuroprotective activity against α-synuclein toxicity in this system.

TABLE 4 SA compounds prevent/inhibit rotenone-induced cell death inA53T-mutant α-synuclein neuroblastoma cells SA # IC₅₀ (μM) Max %Inhibition 52 23.8 64 58 3.5 100 59 3.1 63 60 12.5 33 61 3.6 93 62 3.631 64 2.6 100 67 8.5 44 68 24 61 69 35-75 30 70 5.6 78 72 10.3 39 7335-75 77 93 19.8 100 94 2.3 58 95 3.8 63 96 4.3 61 97 39 42 98 48 56Positive Ref. #2 4.2 72 Positive Ref. #3 10.6 82 Negative Ref. notdetermined 0

Example 39 Compounds of this Invention Directly Inhibit the In VitroConversion of α-Synuclein to β-Sheet Containing Structures

As described above, Thioflavin S histochemistry in α-synucleinexpressing cells was used to detect aggregates containing a high degreeof β-pleated sheet structures that formed in response to rotenonetreatment. Since several compounds were shown to reduce the abundance ofThioflavin S-positive aggregates (Example 3), we sought independentconfirmation that the compounds directly inhibit the conversion ofα-synuclein to β-sheet containing structures by using circular dichroism(CD) spectroscopy. For this purpose, α-synuclein was obtained fromrPeptide as a lyophilized salt in 1 mg aliquots. Buffer components andother solvents were obtained from Sigma as A.C.S. Reagent grade orhigher. Wild-type α-synuclein was dissolved in a buffer containing 9.5mM phosphate, 137 mM sodium chloride and 2.7 mM potassium chloride(phosphate-buffered saline; PBS), and the pH was adjusted to pH 7.4.This solution was then re-lyophilized and dissolved in 0.5 mL deionizedwater at 2 mg/mL (138 μM), and an aliquot taken and diluted to 0.05mg/mL in PBS for CD spectral analysis (t=0, unfolded reference control).In order to induce aggregation, 1 mg/ml α-synuclein (69 μM) wasincubated at 37° C. for 24 hours with shaking (1,300 rpm), either alone,or in the presence of one of the test compounds (at testcompound:α-synuclein molar ratios of 5:1, 1:1, 0.5:1, 0.1:1, 0.05:1,0.01:1). After 24 hours, reactions were diluted 20-fold in PBS and CDspectra for each reaction were acquired on a Jasco J-810spectropolarimeter using a 0.1 cm path length cell. All spectra wererecorded with a step size of 0.1 nm, a bandwidth of 1 nm, and anα-synuclein concentration of 0.05 mg/ml. The spectra were trimmed at theshortest wavelength that still provided a dynode voltage less than 600V.The trimmed spectra were then subjected to a data processing routinebeginning with noise reduction by Fourier transform followed bysubtraction of a blank spectrum (vehicle only without α-synuclein).These blank corrected spectra were then zeroed at 260 nm and the unitsconverted from millidegrees to specific ellipticity.

Percent β-sheet was determined from processed spectra using theellipticity minimum value at approximately 218 nm and referencing to ascale normalized to nearly fully folded and unfolded reference values,consistent with previous reports (Ramirez-Alvarado et al., J. Mol.Biol., 273:898-912, 1997; Andersen et al., J. Am. Chem. Soc.,121:9879-9880, 1999) The fully folded reference value was found byperforming the described calculation on the spectrum of α-synucleinfibrillized for 24 hours (complete fibrilization), and assigning thisdifference the arbitrary value of 100% β-sheet. The unfolded referencewas provided by the spectrum from the same sample at the initial timepoint (t=0) and ascribing the difference found here the arbitrary valueof 0% β-sheet. These percent β-sheet values were then used to providethe respective relative % inhibition of β-sheet induced by the compoundsat given molar ratio of test compound:α-synuclein. For each compound,the % inhibition of β-sheet formation was plotted against the log of theconcentration (mole ratio) of the test compound and, where possible, the50% inhibitory concentration (IC₅₀) was calculated from the doseresponse curve.

First, in order to confirm that α-synuclein is indeed converted to aβ-sheet-rich structure and to establish the timing of this conversion at24 hours in our system, an aliquot of the α-synuclein only incubationmixture (without compounds) was sampled at various time points and CDspectra collected. At 24 hours of incubation, CD analysis revealed alarge abundance of a β-sheet-rich structure, indicated by the pronouncedspecific ellipticity minimum at 218 nm and maximum at 197 nm (notshown). However, when test compounds SA-54, SA-55, SA-57, SA-58, SA-59,SA-61, SA-62, SA-64, SA-67, SA-68, SA-70, SA-72, SA-93, SA-94, SA-95,SA-96, SA-97, SA-98, SA-99 or positive reference compound #1 wereincluded individually in the reaction mixture, at appropriateconcentrations, and the incubation mixture sampled 24 hours later, therewas an absence of the minimum at 218 nm. Instead, a spectrumcharacteristic of random coil was exhibited (not shown). We concludethat these compounds prevent the conversion of natively unfoldedα-synuclein to a β-sheet-rich structure. These results are summarized inTable 5. As a specific example, compound SA-57 resulted in nearlycomplete inhibition when used at test compound:α-synuclein molar ratios≧0.05:1, with a calculated IC₅₀ of 0.026 moles of compound per mole ofα-synuclein (FIG. 5), whereas the negative reference compound did notsignificantly inhibit β-sheet formation at any of the tested molarratios (not shown). Nearly all of the compounds that inhibitedα-synuclein β-sheet formation did so at less than equimolar ratiosrelative to α-synuclein (i.e. IC₅₀ molar ratios <1), although somecompounds (for example, SA-99; IC₅₀=2.08) required higher concentrationsin order to markedly inhibit α-synuclein β-sheet formation (Table 5).Taken together, these results indicate that these SA compounds showpotent inhibition and prevention of α-synuclein aggregation a hallmarkof the synucleinopathies such as Parkinson's disease.

TABLE 5 SA compounds prevent/inhibit β-sheet containing α-synucleinaggregates as assessed by circular dichroism spectroscopy. SA # IC₅₀(mole ratio) Max % Inhibition 54 0.55 100 55 0.055 100 57 0.026 100 580.36 94 59 0.75 99 61 0.78 95 62 0.54 88 64 0.56 100 67 0.41 100 68 0.6100 70 0.25 92 72 0.27 100 93 0.38 91 94 0.22 95 95 0.68 97 96 0.13 10097 not determined 100 98 0.08 100 99 2.08 98 Positive Ref. #1 0.04 100Negative Ref. not determined none detected

Example 40 Compounds Provided Herein Bind with High Affinity toParkinson's Disease α-Synuclein Fibrils

The compounds prepared in the preceding examples were found to bind withhigh affinity to α-synuclein aggregates/fibrils that are found in thehallmark Lewy Bodies of Parkinson's disease. In order to assess relativebinding affinities of the test compounds for aggregated α-synuclein,competition assays were set up with a radiolabeled molecule alreadyknown to bind to α-synuclein fibrils and non-radiolabeled testcompounds. In order to induce its aggregation, α-synuclein was incubatedin phosphate buffered saline (PBS, pH 7.4) at 37° C. for three days withshaking (1,400 rpm). Competitive binding assays were carried out in12×75 mm borosilicate glass tubes. The reaction mixture contained 100 μLof α-synuclein aggregates (0.5-1 μg), [³H] positive reference compound#1 (100-200 nM diluted in PBS) and 50 μL of competing compounds(10⁻⁵-10⁻⁹ M, diluted serially in PBS containing 0.1% bovine serumalbumin) in a final volume of 0.25 ml. Non-specific binding was definedin the presence of cold positive reference compound #1 (50 μM) in thesame assay tubes. The mixture was incubated for 120 min at 37° C., andthe bound and the free radioactivity were separated by vacuum filtrationthrough Whatman GF/B filters using a Brandel M-24R cell harvester,followed by washing with PBS buffer three times. Filters containing thebound [³H] positive reference compound #1 were assayed for radioactivityin a liquid scintillation counter (Beckman LS6500). IC₅₀ values weredetermined by a non-linear, least squares regression analysis.Inhibition constants (Ki) values were calculated using the equation ofCheng and Prusoff (Cheng et al., Biochemical Pharmacology 22:3099-3108,1973) using the observed IC₅₀ of the tested compound, the concentrationof radioligand employed in the assay, and the value for the Kd of theligand (600 nM).

The results from these experiments are reported in Table 6. As anexample, SA-64 binds with high affinity to α-synuclein fibrils (Ki=89nM) but does not show significant binding affinity for the amyloid-βpeptide of Alzheimer's disease (not shown). Similarly, SA-58 and SA-57bind with high affinity to α-synuclein aggregates, with bindingconstants (Ki) of 105 nM and 124 nM, respectively. The increased bindingaffinity (by 4-5-fold) of SA-57 and SA-58, relative to positivereference molecule #1 represents a significant improvement in binding toα-synuclein aggregates for these new molecules. Taken together, theseresults indicate that SA compounds bind to varying degrees to theα-synuclein aggregates, a hallmark of synucleinopathies such asParkinson's disease.

TABLE 6 SA compounds bind to α-synuclein aggregates as measured by an invitro competition binding assay. SA # K_(i) (nM) 52 1260 53 813 54 164055 370 57 124 58 105 59 697 61 3100 62 506 63 283 64 89 66 135 67 466 68697 69 765 70 659 72 270 76 219 78 330 79 537 82 177 83 330 84 639 86224 87 2000 88 970 89 1200 90 1700 94 1100 95 890 96 1350 Positivereference #1 532 negative reference #1 no binding negative reference #2>10000

Example 41 Use of Recombinant Tau Repeat Domain for in Vitro Screeningof Tau Aggregation Inhibitors

During in vitro screening for identification of tau aggregationinhibitors, we found that under the same experimental conditions,formation of paired helical filaments (PHFs) from commercially-purchasedfull-length tau protein (e.g. Tau441; rPeptide) was much slower (>11days) than that from the tau repeat domain (TauRD; containing Q244-E372of Tau441) (≧24 hours). Because of the remarkably short turn-around timeand common aggregation properties, we used TauRD for in vitro drugscreening to identify tau aggregation inhibitors [Barghorn S, Biernat J,and Mandelkow E, Purification of recombinant tau protein and preparationof Alzheimer-paired helical filaments in vitro. Methods Mol Biol, 2005.299: p. 35-51]. Since the TauRD protein is not commercially available,we produced our own protein for this study. A cDNA fragment coding forthe human TauRD (Q244-E372 of Tau441) was cloned into a bacterialexpression vector and the construct was then expressed in E. Coli. Therecombinant TauRD protein was then purified by heat-stability treatmentand cation exchange chromatography as described [Barghorn, et al.,] withminor modifications. Using this method, we achieved a protein yield of10 mg per liter of bacterial culture, with >95% purity. Aggregation andPHF formation of purified TauRD were evaluated and validated byindependent assays including Thio S fluorometry, CD spectroscopy andelectron microscopy (Data not shown). The results consistentlydemonstrate that TauRD (10 μM) is able to form Thio S-positive,β-sheet-containing PHFs when incubated with an equal concentration ofheparin, at 37° C. (with shaking at 800-1000 rpm for day).

Example 42 Identification of Novel Tau Aggregation Inhibitors byThioflavin S Fluorometry Screening

The Thio S fluorometry assay as a primary screening method to identifytau protein aggregation inhibitors from our small molecule library.Aggregated tau fibrils were prepared in the presence of equimolar ratiosof TauRD and heparin (10 μM each) in 20 mM Na-phosphate buffer, pH7.4.The reaction mixture was incubated at 37° C. with shaking (800-1000 rpm)for 22-24 hr (or for 3 days). In the Thio S inhibition assays, testcompounds at 0, 0.1, 1, 10 and 100 μM were added at time 0 into thereaction containing TauRD and heparin. The same reaction mixture (+/−increasing concentrations of compounds) but without TauRD were also setup in parallel to serve as background controls. For all test compoundsbackground fluorescence readings were very low, usually <5% of those ofthe TauRD-containing wells. For each compound, the IC₅₀ was calculatedusing Prism version 5 software (GraphPad Software) by nonlinearregression [(Log [inhibitor] vs. normalized response; variable slope)].In initial screening, 20 test compounds demonstrated a broad range ofactivities for inhibiting tau protein fibril formation: IC₅₀ valuesranged from ˜5 μM to infinity (i.e. no activity at all). The resultssuggested that the inhibitory activities were structure specific. TheThio S screening results are summarized in Table 7 (in which thereactions were incubated for 22 hours).

TABLE 7 SA compounds inhibit tau protein fibril formation as measured byThioflavin S fluorometry. Compounds ThioS (IC₅₀, μM) SA-97 5.20 SA-547.34 SA-95  9.02 ± 4.66 (n = 2) SA-63 10.08 ± 0.56 (n = 2) SA-57 10.24SA-61 12.54 ± 1.24 (n = 2) SA-64 17.61 SA-96 21.21 SA-94 21.44 SA-9924.70 SA-52 32.83 SA-68 33.96 SA-98 78.51 SA-70 117.00 SA-59 211.10SA-72 290.20 SA-67 3941.00 SA-62 no inhibition SA-55 no inhibition SA-60no inhibition

Example 43 Select Compounds Also Inhibit Tau Protein Formation ofβ-Sheet Secondary Structures Characteristic of Neurofibrillary Tanglesas Determined by Circular Dichroism Spectroscopy

CD spectroscopy was also performed to determine each compound's potencyin inhibiting β-sheet secondary structure in TauRD underaggregation-prone conditions. The CD spectroscopy and Thio S assays weretypically analyzed in parallel from the same sample preparation in orderto correlate the results from two independent assays. CD spectra weretaken from the samples containing +/− TauRD with increasingconcentrations of compounds, and collected at 25° C. on a JASCO ModelJ-810 Spectropolarimeter. To determine compound inhibitory potency, weestablished a semi-quantitative scoring system to illustrate TauRDconformational changes on CD spectra explained below. Since CD spectrareflect a total population of secondary structures (including randomcoil, β-sheet, and various intermediate conformers) of TauRD proteinsunder a given condition, the CD scores were established based on (1) theCD spectra derived from TauRD mixtures with different ratios of randomcoil/β-sheet; (2) time-dependent conformational changes of TauRD. CDanalysis revealed that non-aggregated TauRD proteins in solution (attime 0 in the presence of heparin, or at various times of incubation inthe absence of heparin) showed spectra with ellipticity minima near 195nm, characteristic of largely random coil structures (not shown). Incontrast, aggregated and fibrillar TauRD showed spectra with minima near218 nm, characteristic of β-sheet secondary structure (not shown). OurCD analysis studies confirmed some of our compounds that could inhibitformation of tau protein β-sheet structure.

TABLE 8 SA compounds prevent/inhibit β-sheet containing tau protein asassessed by circular dichroism spectroscopy. Compounds CD SA-97 ++ SA-54− SA-95 + SA-63 + SA-57 − SA-61 + SA-64 − SA-96 − SA-94 − SA-99 +SA-52 + SA-68 No data SA-98 No data SA-70 No data SA-59 − SA-72 − SA-67n/t SA-62 n/t SA-55 − SA-60 n/t

Table 8 summarizes data from aggregated TauRD proteins in the presenceof various SA-compounds where the CD score of ‘−’ indicates that the CDspectrum is similar to that of no compound controls with ellipticityminima at 218 nm (β-sheet) and ‘+’ indicates that the Minima remained at218 nm but with a reduced magnitude (intermediate conformers) and ‘++’indicates that the minima shifted to between 195-218 nm (intermediateconformers)

Example 44 Compositions of Compounds Provided Herein

The compounds provided herein, as mentioned previously, are desirablyadministered in the form of pharmaceutical compositions. Suitablepharmaceutical compositions, and the method of preparing them, arewell-known to persons of ordinary skill in the art and are described insuch treatises as Remington: The Science and Practice of Pharmacy, A.Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins,Philadelphia, Pa. Representative compositions are as follows.

Oral Tablet Formulation

% w/w Compound provided herein 10.0 Magnesium stearate 0.5 Starch 2.0Hydroxypropylmethylcellulose 1.0 Microcrystalline cellulose 86.5

The ingredients are mixed to homogeneity, then granulated with the aidof water, and the granulates dried. The granulate is then compressedinto tablets sized to give a suitable dose of the compound. The tabletis optionally coated by applying a suspension of a film forming agent(e.g. hydroxypropylmethylcellulose), pigment (e.g. titanium dioxide),and plasticizer (e.g. diethyl phthalate), and drying the film byevaporation of the solvent. The film coat may comprise, for example,2-6% of the tablet weight.

Oral Capsule Formulation

The granulate from the previous section of this Example is filled intohard gelatin capsules of a size suitable to the intended dose. Thecapsule is banded for sealing, if desired.

Softgel Formulation

A softgel formulation is prepared as follows:

% w/w Compound provided herein 20.0 Polyethylene glycol 400 80.0

The compound is dissolved or dispersed in the polyethylene glycol, and athickening agent added if required. A quantity of the formulationsufficient to provide the desired dose of the compound is then filledinto softgels.

Parenteral Formulation

A parenteral formulation is prepared as follows:

% w/w Compound provided herein 1.0 Normal saline 99.0

The compound is dissolved in the saline, and the resulting solution issterilized and filled into vials, ampoules, and prefilled syringes, asappropriate.

Controlled-Release Oral Formulation

A sustained release formulation may be prepared by the method of U.S.Pat. No. 4,710,384, as follows:

One kilogram of a compound provided herein is coated in a modifiedUni-Glatt powder coater with Dow Type 10 ethyl cellulose. The sprayingsolution is an 8% solution of the ethyl cellulose in 90% acetone to 10%ethanol. Castor oil is added as plasticizer in an amount equal to 20% ofthe ethyl cellulose present. The spraying conditions are as follows: 1)speed, 1 liter/hour; 2) flap, 10-15%; 3) inlet temperature, 50° C., 4)outlet temperature, 30° C., 5) percent of coating, 17%. The coatedcompound is sieved to particle sizes between 74 and 210 microns.Attention is paid to ensure a good mix of particles of different sizeswithin that range. Four hundred mg of the coated particles are mixedwith 100 mg of starch and the mixture is compressed in a hand press to1.5 tons to produce a 500 mg controlled release tablet.

The claimed subject matter is not limited in scope by the specificembodiments described herein. Indeed, various modifications of thespecific embodiments in addition to those described will become apparentto those skilled in the art from the foregoing descriptions. Suchmodifications are intended to fall within the scope of the appendedclaims. Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

1. A compound selected from the group consisting of:


2. A method of disrupting or inhibiting the formation, deposition,accumulation, or persistence of tau fibrils and/or aggregates,comprising administering a therapeutically effective amount of thecompounds of claim
 1. 3. The method of claim 2, where the compoundadministered is in an amount between 0.1 mg/Kg/day and 1000 mg/Kg/day.4. The method of claim 2, where the compound is administered in anamount between 1 mg/Kg/day and 100 mg/Kg/day.
 5. The method of claim 2,where amount of compound administered is in an amount between 10mg/Kg/day and 100 mg/Kg/day.
 6. A method resulting in neuroprotectionfrom a tauopathy in a mammal comprising the step of administrating atherapeutically effective amount of a compound of claim
 1. 7. An articleof manufacture, comprising packaging material, the compound of claim 1,or a pharmaceutically acceptable salt thereof, contained withinpackaging material, which is used for treating the formation,deposition, accumulation, or persistence of tau fibrils and/oraggregates, and a label that indicates that the compound orpharmaceutically acceptable salt thereof is used for treating theformation, deposition, accumulation, or persistence of tau fibrilsand/or aggregates.
 8. A composition comprising the compound of claim 1and at least one pharmaceutically acceptable excipient.
 9. A method oftreating a tauopathy in a mammal comprising the step of administrating atherapeutically effective amount of a compound of claim
 1. 10. Themethod of claim 9 wherein the tauopathy is selected from the groupconsisting of Alzheimer's disease with neuronal and glial taudeposition, Pick's disease, progressive supranuclear palsy, corticobasaldegeneration, familial frontotemporal dementia/Parkinsonism linked tochromosome 17, amyotrophic lateral sclerosis/Parkinsonism-dementiacomplex, argyrophilic grain dementia, dementia pugilistic, diffuseneurofibrillary tangles with calcification, progressive subcorticalgliosis and tangle only dementia.
 11. The method of claim 9, where thecompound administered is in an amount between 0.1 mg/Kg/day and 1000mg/Kg/day.
 12. The method of claim 9, where the compound is administeredin an amount between 1 mg/Kg/day and 100 mg/Kg/day.
 13. The method ofclaim 9, where amount of compound administered is in an amount between10 mg/Kg/day and 100 mg/Kg/day.